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EGF receptor (EGFR) signaling in human cancers elicits changes in protein-expression patterns that are crucial for potentiating tumor growth. Identifying those proteins with expression regulated by the EGFR and determining how they contribute to malignancy is fundamental for the development of more effective strategies to treat cancer. Here, we show that tissue transglutaminase (tTG) is one such protein. EGF up-regulates tTG expression in human breast-cancer cells, and knock-downs of tTG or the treatment of breast cancer cells with a tTG inhibitor blocks their EGF-stimulated anchorage-independent growth. We further show that the combined actions of Ras and Cdc42, leading to the activation of PI 3kinase and NF[kappa]B, provide a mechanism by which EGF can up-regulate tTG in breast-cancer cells. Moreover, overexpression of wild-type tTG, but not its transamidation-defective counterpart, fully mimics the growth advantages afforded by EGF to these cancer cells. Surprisingly, the tTG-promoted growth of breast-cancer cells is dependent on its ability to activate the Src tyrosine kinase as an outcome of a complex formed between tTG and the breast-cancer marker and intermediate filament protein keratin-19. These findings identify tTG as a key participant in an EGFR/Src-signaling pathway in breast-cancer cells and a potential target for inhibiting EGFR-promoted tumor progression. breast cancer | keratin 19 doi/ 10.1073/pnas.0907907107

Our recent studies showed that transglutaminase-1 (TGase-1) is uniquely expressed in mouse renal proximal tubular cells (RPTC) and mediates cell proliferation. In this study, we investigated the role of TGase-1 in cell survival and the survival signaling pathways regulated by TGase-1 in RPTC following oxidant injury. Exposure of RPTC to hydrogen peroxide ([H.sub.2][O.sub.2]) resulted in apoptosis and an increase in TGase activity. Inhibition of TGase activity with monodansylcadervine (MDC), a TGase inhibitor, or knockdown of TGase-1 with small interference (si)RNA enhanced apoptosis and decreased cell survival in [H.sub.2][O.sub.2]-treated RPTC. Conversely, overexpression of TGase-1 rendered RPTC more resistant to [H.sub.2][O.sub.2] toxicity and MDC treatment blocked this response. Concurrent with RPTC apoptosis, phosphorylation of AKT, signal transducer and activator of transcription-3 (STAT3), and glucogen synthase kinase-3[beta] (GSK-3[beta]) were observed. Pretreatment of cells with MDC or TGase-1 siRNA inhibited phosphorylation of all these molecules. Inhibition of either the AKT or STAT3 pathway potentiated [H.sub.2][O.sub.2]-induced cell death and increased GSK-3[beta] activity by dephosphorylation at serine 9. Furthermore, treatment with GSK-3[beta] inhibitors reduced [H.sub.2][O.sub.2]-induced apoptosis and abolished the death-promoting effect of AKT and STAT3 inhibition. Therefore, we have identified TGase-1 as a novel survival factor in renal epithelial cells and it contributes to cell survival through activation of the AKT and STAT3 signaling pathways following oxidant injury. renal proximal tubular cells doi: 10.1152/ajprenal.00251.2009.

Gut 2003;52:1567-1571 Background: IgA serum autoantibodies against tissue transglutaminase (tTG) have an established diagnostic value in coeliac disease, and high efficacy tests are widely available for their detection. However, serological [...]

Proteinaceous aggregates containing [alpha]-synuclein represent a feature of neurodegenerative disorders such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Despite extensive research, the mechanisms underlying [alpha]-synuclein aggregation remain elusive. Previously, tissue transglutaminase (tTGase) was found to contribute to the generation of aggregates by cross-linking pathogenic substrate proteins in Huntington's and Alzheimer's diseases. In this article, the role of tTGase in the formation of [alpha]-synuclein aggregates was investigated. Purified tTGase catalyzed [alpha]-synuclein cross-linking, leading to the formation of high molecular weight aggregates in vitro, and overexpression of tTGase resulted in the formation of detergent-insoluble [alpha]-synuclein aggregates in cellular models. Immunocytochemical studies demonstrated the presence of [alpha]-synuclein-positive cytoplasmic inclusions in 8% of tTGase-expressing cells. The formation of these aggregates was significantly augmented by the calcium ionophore A23187 and prevented by the inhibitor cystamine. Immunohistochemical studies on postmortem brain tissue confirmed the presence of transglutaminase-catalyzed [epsilon]([gamma]-glutamyl)-lysine cross-links in the halo of Lewy bodies in Parkinson's disease and dementia with Lewy bodies, colocalizing with [alpha]-synuclein. These findings, taken together, suggest that tTGase activity leads to [alpha]-synuclein aggregation to form Lewy bodies and perhaps contributes to neurodegeneration.

Background and aims: Coeliac disease (CD) is a multifactorial disarder which has an autoimmune camponent characterised by the occurrence of disease specific autoreactive antibodies against the enzyme tissue transglutaminase (tTG). [...]

Transglutaminase 2 (TG2) is a distinctive member of the family of [Ca.sup.2+]-dependent enzymes recognized mostly by their abilities to catalyze the posttranslational crosslinking of proteins. TG2 uniquely binds and hydrolyzes GTP; binding GTP inhibits its crosslinking activity but allows it to function in signal transduction (hence the [G.sub.h] designation). The core domain of TG2 (residues 139-471, rat) comprises the papain-like catalytic triad and the GTP-binding domain (residues 159-173) and contains almost all of the conserved tryptophans of the protein. Examining point mutations at Trp positions 180, 241, 278, 332, and 337 showed that, upon binding 2'-(or 3')-O-(N-methylanthraniloyl)GTP (mantGTP), the Phe-332 mutant was the weakest (35% less than wild type) in resonance energy transfer from the protein ([[lambda].sub.exc, max] = 290 nm) to the mant fluorophore ([[lambda].sub.em] = 444 nm) and had a reduced affinity for mantGTP. Trp-332, situated near the catalytic center and the nucleotide-binding area of TG2, may be part of the allosteric relay machinery that transmits negative effector signals from nucleotide binding to the active center of TG2. A most important observation was that, whereas no enzyme activity could be detected when Trp-241 was replaced with Ala or Gin, partial preservation of catalytic activity was seen with substitutions by Tyr > Phe > His. The results indicate that Trp-241 is essential for catalysis, possibly by stabilizing the transition states by H-bonding, quadrupole-ion, or van der Waals interactions. This contrasts with the evolutionarily related papain family of cysteine proteases, which uses Gln-19 (papain) for stabilizing the transition state.

Tissue transglutaminase (TG) is a [Ca.sup.2+]-dependent acyltransferase with roles in cellular differentiation, apoptosis, and other biological functions. In addition to being a transamidase, TG undergoes a GTP-binding/GTPase cycle even though it lacks any obvious sequence similarity with canonical GTP-binding (G) proteins. Guanine nucleotide binding and [Ca.sup.2+] concentration reciprocally regulate TG's transamidation activity, with nucleotide binding being the negative regulator. Here we report the x-ray structure determined to 2.8-[Angstrom] resolution of human TG complexed with GDP. Although the transamidation active site is similar to those of other known transglutaminases, the guanine nucleotide-binding site of TG differs markedly from other G proteins. The structure suggests a structural basis for the negative regulation of transamidation activity by bound nucleotide, and the positive regulation of transamidation by [Ca.sup.2+].

Research has been conducted on the Celiac Sprue, an autoimmune disorder of the human small intestine. The catalytic and allosteric regulatory properties of the tissue transglutaminase pertaining to the Celiac Sprue pathogenesis have been investigated and the results are discussed.

Research has been conducted on the guinea pig liver transglutaminase. The thiolester acyl enzyme intermediate deacylation conducted via catalytic aminolysis has been investigated in the presence of primary amines.

The expression of transglutaminase mRNA in rat small intestinal crypt cells (IEC-6 cell line) and human colon carcinoma cells (Caco-2 cell lines) was measured after alpha-difluoromethylornithine-induced cellular polyamine depletion. Results reveal that IEC-6 cell lines requires polyamines in the transcription if transglutaminase mRNA but these polyamines inhibit transglutaminase mRNA synthesis in Caco-2 cells. There are different mechanisms of transglutaminase mRNA transcription regulation in these cell lines.

A lot of questions still remain unanswered in the searching for the role that transglutaminase (TG) plays in the progression of neurodegenerative diseases including Alzheimer's. It was D.J. Selkoe and his colleagues who first suggested that TG interacting with neurofilaments might be involved in neurodegenerative diseases. There are now nontoxic active center-directed inhibitors of TG available in the market including Zn2+. New knowledge on the subject could be gained on studies using these inhibitors on AD and SCA1 transgenic mice.

The [alpha.sub.1] adrenergic receptors activate a phospholipase C enzyme by coupling to members of the large molecular size (approximately 74 to 80 kilodaltons) [G[alpha.sub.h] family of guanosine triphosphate (GTP)-binding proteins. Rat liver [G[alpha.sub.h] is now shown to be a tissue transglutaminasetype II (TGase II). The transglutaminase activity of rat liver TGase II expressed in COS-1 cells was inhibited by the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) or by [alpha.sub.1]-adrenergic receptor activation. Rat liver TGase II also mediated [alpha.sub.1]-adrenergic receptor stimulation of phospholipase C activity. Thus, [G[alpha.sub.h] represents a new class of GTP-binding proteins that participate in receptor signaling and may be a component of a complex regulatory network in which receptor-stimulated GTP binding switches the function of [G[alpha.sub.h] from transglutamination to receptor signaling., We have shown previously that a GTP-binding protein, termed [G.sub.h] copurifies with rat liver [alpha.sub.1]-adrenergic receptors in a ternary complex containing [alpha.sub.1]-agonist, the receptor, and [G.sub.h][1]. De novo purification of [...]

Complex formation between the human erythrocyte (RBC) transglutaminase and fibronectin or its fragments was examined by immunoanalytical procedures and by fluorescence polarization. It was shown that a 42-kDa gelatin-binding structure displays a high specificity for binding to the human RBC enzyme. This structure is sequentially composed of a type I, followed by two type II and three type I motifs.

Transglutaminase catalyzes the Ca2+ -dependent transfer of the acyl group in glutamine to various acyl acceptors. The distribution of tissue transglutaminase in skeletal tissues was investigated to determine the role of the enzyme in physiological cross-linking of the extracellular matrix. The results showed that transglutaminase expression is correlated with the terminal differentiation of chondrocytes. Type II collagen and osteonectin served as the primary substrates of the enzyme.

The effects of vitamin A nutritional status on the levels of expression of retinoic acid nuclear receptors (RAR), and the retinoic acid-responsive gene, tissue transglutaminase, were determined in rats. Weanling male Sprague-Dawley rats fed a vitamin A-deficient diet for approximately 7 wk developed vitamin A deficiency, as confirmed by the depletion of liver retinol and retinyl palmitate. Controls were fed the same diet supplemented with 24 mg/kg retinyl acetate. The levels of expression of RAR-beta mRNA were approximately 80% lower in bladder, brain, liver, lung and trachea and those of RAR-gamma mRNA were approximately 50% lower in bladder, lung and trachea of rats fed the vitamin A-deficient diet than in controls. The levels of expression of RAR-alpha mRNA were approximately 90% lower in brain and approximately 30% greater in liver, kidney, intestine and lung of rats fed the vitamin A-deficient diet.

Balb-C 3T3 fibroblasts were transfected with a constitutive tissue transglutaminase (TT) expression plasmid to investigate the role of TT in physiological systems. The results showed that TT-transfected cells exhibited very flattened and extended morphology. Membrane blebbing and cellular fragmentation were observed in cells with the highest levels of TT expression. These results indicate that TT has an important role in alterations of the morphology and adhesiveness of cells.

Short peptides containing transglutaminase-reactive glutamine residue and an easily recognizable dansyl reporter group were used toidentify the lysine-carrying protein substrates of the enzyme in bovine lens. The peptides inhibited the transglutaminase-catalyzed cross-linking of proteinsand became incorporated into the substrate of the enzyme. The study revealed the transglutaminase carboxyl-terminal domain, EKPAVTAAPKK to be the prominent lysine-donor sequence in bovine lens.

Research has been conducted on secretion of active-form transglutaminase, previously observed in Corynebacterium glutamicum. The authors repor t the study of SAM-P45 cleavage site in the C-terminal region of the prodomain carried out via the use of site-directed mutagenesis in order to secrete nativ e-type Streptoverticillium mobaraense?transglutaminase.

The cause of Huntington's disease (HD) is a pathological expansion of the polyglutamine domain within the [NH.sub.2]-terminal region of huntingtin. Neuronal intranuclear inclusions and cytoplasmic aggregates composed of the mutant huntingtin within certain neuronal populations are a characteristic hallmark of HD. Because in vitro expanded polyglutamine repeats are glutaminyl-donor substrates of tissue transglutaminase (tTG), it has been hypothesized that tTG may contribute to the formation of these aggregates in HD. Therefore, it is of fundamental importance to establish whether tTG plays a significant role in the formation of mutant huntingtin aggregates in the cell. Human neuroblastoma SH-SY5Y cells were stably transfected with truncated [NH.sub.2]-terminal huntingtin constructs containing 18 (wild type) or 82 (mutant) glutamines. In the cells expressing the mutant truncated huntingtin construct, numerous SDS-resistant aggregates were present in the cytoplasm and nucleus. Even though numerous aggregates were present in the mutant huntingtin-expressing cells, tTG did not coprecipitate with mutant truncated huntingtin. Further, tTG was totally excluded from the aggregates, and significantly increasing tTG expression had no effect on the number of aggregates or their intracellular localization (cytoplasm or nucleus). When a YFP-tagged mutant truncated huntingtin construct was transiently transfected into cells that express no detectable tTG due to stable transfection with a tTG antisense construct, there was extensive aggregate formation. These findings clearly demonstrate that tTG is not required for aggregate formation, and does not facilitate the process of aggregate formation. Therefore, in HD, as well as in other polyglutamine diseases, tTG is unlikely to play a role in the formation of aggregates. Key words: Huntington's disease * inclusions * polyglutamine * polar zipper * isopeptide bond

Johnson, K. A., L. Hessle, S, Vaingankar, C. Wennberg, S. Mauro, S. Narisawa, J. W. Goding, K. Sano, J. L. Millan, and R. Terkeltaub. Osteoblast tissue-nonspecific alkaline phosphatase antagonizes and regulates PC-1. Am J Physiol Regulatory Integrative Comp Physiol 279: R1365-R1377, 2000.-- Tissue-nonspecific alkaline phosphatase (TNAP) is essential for bone matrix mineralization, but the central mechanism for TNAP action remains undefined. We observed that ATP-dependent [sup.45]Ca precipitation was decreased in calvarial osteoblast matrix vesicle (MV) fractions from TNAP-/-mice, a model of infantile hypophosphatasia. Because TNAP hydrolyzes the mineralization inhibitor inorganic pyrophosphate ([PP.sub.i]), we assessed phosphodiesterase nucleotide pyrophosphatase (PDNP/NTPPPH) activity, which hydrolyzes ATP to generate [PP.sub.i]. Plasma cell membrane glycoprotein-1 (PC-1), but not the isozyme B10 (also called PDNP3) colocalized with TNAP in osteoblast MV fractions and pericellular matrix. PC-1 but not B10 increased MV fraction [PP.sub.i] and inhibited [sup.45]Ca precipitation by MVs. TNAP directly antagonized inhibition by PC-1 of MV-mediated [sup.45]Ca precipitation. Furthermore, the [PP.sub.i] content of MV fractions was greater in cultured TNAP-/- than TNAP+/+ calvarial osteoblasts. Paradoxically, transfection with wild-type TNAP significantly increased osteoblast MV fraction NTPPPH. Specific activity of NTPPPH also was twofold greater in MV fractions of osteoblasts from TNAP+/+ mice relative to TNAP-/-mice. Thus TNAP attenuates PC-1/NTPPPH-induced [PP.sub.i] generation that would otherwise inhibit MV-mediated mineralization. TNAP also paradoxically regulates PC-1 expression and NTPPPH activity in osteoblasts. inorganic pyrophosphate; hypophosphatasia; PDNP3; B10; transglutaminase

GTP is known to be a potent inhibitor of the protein crosslinking activity of transglutaminase (TG), probably the most abundant G protein in the human red cell. Nucleotide binding to TG was examined by fluorescence spectroscopy and anisotropy in mixtures of TG with methylanthraniloyl analogs of GTP and GDP. A characteristic feature was the appearance of a major energy transfer band ([[Lambda].sub.exc, max] = 290 nm, [[Lambda].sub.em] = 444 nm) from protein tryptophans to the bound nucleotides. Quenching of the bound fluorophore ([[Lambda].sub.exc] = 360 nm, [[Lambda].sub.em]= 444 nm) by acrylamide was barely different from that of free ligand. However, major changes were observed in anisotropy, which was used to demonstrate a facile exchange between bound and free nucleotides and to evaluate affinity constants for the binding of methylanthraniloyl GTP and GDP to TG.

The protein cross-linking enzyme tissue transglutaminase binds in vitro with high affinity to fibronectin via its 42-kD gelatin-binding domain. Here we report that cell surface transglutaminase mediates adhesion and spreading of cells on the 42-kD fibronectin fragment, which lacks integrin-binding motifs. Overexpression of tissue transglutaminase increases its amount on the cell surface, enhances adhesion and spreading on fibronectin and its 42-kD fragment, enlarges focal adhesions, and amplifes adhesion-dependent phosphorylation of focal adhesion kinase. These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII. Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins. Transglutaminase interacts directly with multiple integrins of [Beta]1 and [Beta]3 subfamilies, but not with [Beta]2 integrins. Complexes of transglutaminase with integrins are formed inside the cell during biosynthesis and accumulate on the surface and in focal adhesions. Together our results demonstrate that tissue transglutaminase mediates the interaction of integrins with fibronectin, thereby acting as an integrin-associated coreceptor to promote cell adhesion and spreading. Key words: adhesion * integrins * tissue transglutaminase * fibronectin * focal adhesions

The inositol phosphate hydrolyzing activity of human phospholipase C[Delta]1 (PLC[Delta]I) is markedly inhibited when the enzyme is coexpressed with the human heart [G.sub.h]/transglutaminase (TG) in human embryonic kidney cells. Because the cotransfection does not affect the amount of PLC[Delta]I in the cells, the depression of phospholipase activity probably is a result of a direct interaction between the two proteins. An ELISA procedure was employed to document the associations of purified TG preparations from a variety of tissues (human red cells, rabbit lens, guinea pig liver) with PLC[Delta]1. Nucleotides (GTP [is greater than] GDP [is greater than] ATP [is greater than] GMP = ADP, in order of decreasing efficiency) interfered with the formation of the PLC[Delta]1:TG complex. A conformational change in the TG partner, occurring with nucleotide binding, is thought to be responsible for dissociating the two proteins. The structural rearrangement produces a remarkable shift in the anodic mobility of TG in electrophoresis: [TG.sub.slow] + GTP ?? [[TG:GTP].sub.fast]. Altogether, our findings indicate that GTP controls PLC[Delta]I activity by releasing this protein from an inhibitory association with [G.sub.h]/transglutaminase.

A molecular study has shown for the first time that transglutaminase 2 (TGase 2) overexpression induced by transforming growth factor β (TGF-β) is a key step in the development of [...]

Aims: Tissue transglutaminase (tTG) is a major autoantigen recognised by IgA anti-endomysial antibodies (IgA EMA). Enzyme linked immunosorbent assays (ELISA) for IgA anti-tissue transglutaminase antibodies (IgA tTG) hove therefore been [...]