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Loss of protein kinase Cd (PKCd) activity renders cells resistant to DNA damaging agents, including irradiation; however, the mechanism(s) underlying resistance is poorly understood. Here, we have asked if metabolic reprogramming by PKCd contributes to radioprotection. Analysis of global metabolomics showed that depletion of PKCd affects metabolic pathways that control energy production and antioxidant, nucleotide, and amino acid biosynthesis. Increased NADPH and nucleotide production in PKCd-depleted cells is associated with upregulation of the pentose phosphate pathway (PPP) as evidenced by increased activation of G6PD and an increase in the nucleotide precursor, 5-phosphoribosyl-1-pyrophosphate. Stable isotope tracing with U-[13C6] glucose showed reduced utilization of glucose for glycolysis in PKCd-depleted cells and no increase in U-[13C6] glucose incorporation into purines or pyrimidines. In contrast, isotope tracing with [13C5, 15N2] glutamine showed increased utilization of glutamine for synthesis of nucleotides, glutathione, and tricarboxylic acid intermediates and increased incorporation of labeled glutamine into pyruvate and lactate. Using a glycolytic rate assay, we confirmed that anaerobic glycolysis is increased in PKCd-depleted cells; this was accompanied by a reduction in oxidative phosphorylation, as assayed using a mitochondrial stress assay. Importantly, pretreatment of cells with specific inhibitors of the PPP or glutaminase prior to irradiation reversed radioprotection in PKCd-depleted cells, indicating that these cells have acquired codependency on the PPP and glutamine for survival. Our studies demonstrate that metabolic reprogramming to increase utilization of glutamine and nucleotide synthesis contributes to radioprotection in the context of PKCd inhibition. [ABSTRACT FROM AUTHOR]

Ovarian cancer (OC) is the most lethal gynecological cancer. OC cells have high proliferative capacity, are invasive, resist apoptosis, and tumors often display rearrangement of extracellular matrix (ECM) components, contributing to accelerated tumor progression. The multifunctional protein tissue transglutaminase (TG2) is known to be secreted in the tumor microenvironment, where it interacts with fibronectin (FN) and the cell surface receptor integrin β1. However, the mechanistic role of TG2 in cancer cell proliferation is unknown. Here, we demonstrate that TG2 directly interacts with and facilitates the phosphorylation and activation of the integrin effector protein integrin-linked kinase (ILK) at Ser246. We show that TG2 and p-Ser246-ILK form a complex that is detectable in patientderived OC primary cells grown on FN-coated slides. In addition, we show that coexpression of TGM2 and ILK correlates with poor clinical outcome. Mechanistically, we demonstrate that TG2-mediated ILK activation causes phosphorylation of glycogen synthase kinase-3α/β, allowing β-catenin nuclear translocation and transcriptional activity. Furthermore, inhibition of TG2 and ILK using small molecules, neutralizing antibodies, or shRNA-mediated knockdown blocks cell adhesion to the FN matrix, as well as the Wnt receptor response to the Wnt-3A ligand, and ultimately, cell adhesion, growth, and migration. In conclusion, we demonstrate that TG2 directly interacts with and activates ILK in OC cells and tumors and define a new mechanism that links ECM cues with β-catenin signaling in OC. These results suggest a central role of TG2-FN-integrin clusters in ECM rearrangement and indicate that downstream effector ILK may represent a potential new therapeutic target in OC. [ABSTRACT FROM AUTHOR]

Lipids in complex, protein-enriched films at air/liquid interfaces reduce surface tension. In the absence of this benefit, the light refracting and immunoprotective tear film on eyes would collapse. Premature collapse, coupled with chronic inflammation compromising visual acuity, is a hallmark of dry eye disease affecting 7 to 10% of individuals worldwide. Although collapse seems independent of mutation (unlike newborn lung alveoli), selective proteome and possible lipidome changes have been noted. These include elevated tissue transglutaminase and consequent inactivation through C-terminal cross-linking of the tear mitogen lacritin, leading to significant loss of lacritin monomer. Lacritin monomer restores homeostasis via autophagy and mitochondrial fusion and promotes basal tearing. Here, we discover that lacritin monomer C-terminal processing, inclusive of cysteine, serine, and metalloproteinase activity, generates cationic amphipathic a-helical proteoforms. Such proteoforms (using synthetic peptide surrogates) act like alveolar surfactant proteins to rapidly bind and stabilize the tear lipid layer. Immunodepletion of C- but not N-terminal proteoforms nor intact lacritin, from normal human tears promotes loss of stability akin to human dry eye tears. Stability of these and dry eye tears is rescuable with C- but not N-terminal proteoforms. Repeated topical application in rabbits reveals a proteoform turnover time of 7 to 33 h with gradual loss from human tear lipid that retains bioactivity without further processing. Thus, the processed C-terminus of lacritin that is deficient or absent in dry eye tears appears to play a key role in preventing tear film collapse and as a natural slow release mechanism that restores epithelial homeostasis. [ABSTRACT FROM AUTHOR]

During autophagy, LC3 and GABARAP proteins become covalently attached to phosphatidylethanolamine on the growing autophagosome. This attachment is also reversible. Deconjugation (or delipidation) involves the proteolytic cleavage of an isopeptide bond between LC3 or GABARAP and the phosphatidylethanolamine headgroup. This cleavage is carried about by the ATG4 family of proteases (ATG4A, B, C, and D). Many studies have established that ATG4B is the most active of these proteases and is sufficient for autophagy progression in simple cells. Here we examined the second most active protease, ATG4A, to map out key regulatory motifs on the protein and to establish its activity in cells. We utilized fully in vitro reconstitution systems in which we controlled the attachment of LC3/GABARAP members and discovered a role for a C-terminal LC3-interacting region on ATG4A in regulating its access to LC3/GABARAP. We then used a gene-edited cell line in which all four ATG4 proteases have been knocked out to establish that ATG4A is insufficient to support autophagy and is unable to support GABARAP proteins removal from the membrane. As a result, GABARAP proteins accumulate on membranes other than mature autophagosomes. These results suggest that to support efficient production and consumption of autophagosomes, additional factors are essential including possibly ATG4B itself or one of its proteolytic products in the LC3 family. [ABSTRACT FROM AUTHOR]

AdhesionGprotein-coupled receptors (aGPCRs) represent a distinct family of GPCRs that regulate several developmental and physiological processes. Most aGPCRs undergo GPCR autoproteolysis-inducing domain-mediated protein cleavage, which produces a cryptic tethered agonist (termed Stachel (stinger)), and cleavage-dependent and -independent aGPCR signaling mechanisms have been described. aGPCR G1 (ADGRG1 or G protein-coupled receptor 56 (GPR56)) has pleiotropic functions in the development of multiple organ systems, which has broad implications for human diseases. To date, two natural GPR56 ligands, collagen III and tissue transglutaminase (TG2), and one small-molecule agonist, 3-α-acetoxydihydrodeoxygedunin (3-α-DOG), have been identified, in addition to a synthetic peptide, P19, that contains seven amino acids of the native Stachel sequence. However, the mechanisms by which these natural and small-molecule agonists signal through GPR56 remain unknown. Here we engineered a noncleavable receptor variant that retains signaling competence via the P19 peptide. We demonstrate that both natural and small-molecule agonists can activate only cleaved GPR56. Interestingly, TG2 required both receptor cleavage and the presence of a matrix protein, laminin, to activate GPR56, whereas collagen III and 3-α-DOG signaled without any cofactors. On the other hand, both TG2/laminin and collagen III activate the receptor by dissociating the N-terminal fragment from its C-terminal fragment, enabling activation by the Stachel sequence, whereas P19 and 3-α-DOG initiate downstream signaling without disengaging the N-terminal fragment from its C-terminal fragment. These findings deepen our understanding of how GPR56 signals via natural ligands, and a small-molecule agonist may be broadly applicable to other aGPCR family members. [ABSTRACT FROM AUTHOR]

High-mobility group box 1 (HMGB1) is a chromatin-associated protein that, in response to stress or injury, translocates from the nucleus to the extracellular milieu, where it functions as an alarmin. HMGB1's function is in part determined by the complexes (HMGB1c) it forms with other molecules. However, structural modifications in the HMGB1 polypeptide that may regulate HMGB1c formation have not been previously described. In this report, we observed high-molecular weight, denaturing- resistant HMGB1c in the plasma and peripheral blood mononuclear cells of individuals with systemic lupus erythematosus (SLE) and, to a much lesser extent, in healthy subjects. Differential HMGB1c levels were also detected in mouse tissues and cultured cells, in which these complexes were induced by endotoxin or the immunological adjuvant alum. Of note, we found that HMGB1c formation is catalyzed by the protein- cross-linking enzyme transglutaminase-2 (TG2). Cross-link site mapping and MS analysis revealed that HMGB1 can be crosslinked to TG2 as well as a number of additional proteins, including human autoantigens. These findings have significant functional implications for studies of cellular stress responses and innate immunity in SLE and other autoimmune disease. [ABSTRACT FROM AUTHOR]

Candida albicans is an opportunistic fungus that is part of the normal microflora commonly found in the human digestive tract and the normal mucosa or skin of healthy individuals. However, in immunocompromised individuals, it becomes a serious health concern and a threat to their lives and is ranked as the leading fungal infection in humans worldwide. As existing treatments for this infection are non-specific or under threat of developing resistance, there is a dire necessity to find new targets for designing specific drugs to defeat this fungus. Some authors reported the presence of the transglutaminase activity in Candida and Saccharomyces, but its identity remains unknown. We report here the phenotypic effects produced by the inhibition of transglutaminase enzymatic activity with cystamine, including growth inhibition of yeast cells, induction of autophagy in response to damage caused by cystamine, alteration of the normal yeast division pattern, changes in cell wall, and inhibition of the yeast-to-mycelium transition. The latter phenomenon was also observed in the C. albicans ATCC 26555 strain. Growth inhibition by cystamine was also determined in other Candida strains, demonstrating the importance of transglutaminase in these species. Finally, we identified enolase 1 as the cell wall protein responsible for TGase activity. After studying the inhibition of enzymatic activities with anti-CaEno1 antibodies and through bioinformatics studies, we suggest that the enolase and transglutaminase catalytic sites are localized in different domains of the protein. The aforementioned data indicate that TGase/Eno1 is a putative target for designing new drugs to control C. albicans infection. [ABSTRACT FROM AUTHOR]

Transglutaminase 2 (TG2) is a ubiquitously expressed, intracellular as well as extracellular protein with multiple modes of post-translational regulation, including an allosteric disulfide bond between Cys-370-Cys-371 that renders the enzyme inactive in the extracellular matrix. Although recent studies have established that extracellular TG2 is switched "on" by the redox cofactor protein thioredoxin-1 (TRX), it is unclear how TG2 is switched "off." Here, we demonstrate that TG2 oxidation by small-molecule biological oxidants, including glutathione, cystine, and hydrogen peroxide, is unlikely to be the inactivation mechanism. Instead, endoplasmic reticulum (ER)-resident protein 57 (ERp57), a protein in the ER that promotes folding of nascent proteins and is also present in the extracellular environment, has the cellular and biochemical characteristics for inactivating TG2. We found that ERp57 colocalizes with extracellular TG2 in cultured human umbilical vein endothelial cells (HUVECs). ERp57 oxidized TG2 with a rate constant that was 400-2000-fold higher than those of the aforementioned small molecule oxidants. Moreover, its specificity for TG2 was also markedly higher than those of other secreted redox proteins, including protein disulfide isomerase (PDI), ERp72, TRX, and quiescin sulfhydryl oxidase 1 (QSOX1). Lastly, siRNA-mediated ERp57 knockdown in HUVECs increased TG2-catalyzed transamidation in the extracellular environment. We conclude that, to the best of our knowledge, the disulfide bond switch in human TG2 represents the first example of a post-translational redox regulatory mechanism that is reversibly and allosterically modulated by two distinct proteins (ERp57 and TRX). [ABSTRACT FROM AUTHOR]

Microbial transglutaminases (MTGs) catalyze the formation of Gln-Lys isopeptide bonds and are widely used for the crosslinking of proteins and peptides in food and biotechnological applications (e.g. to improve the texture of protein-rich foods or in generating antibody-drug conjugates). Currently used MTGs have low substrate specificity, impeding their biotechnological use as enzymes that do not cross-react with nontarget substrates (i.e. as bio-orthogonal labeling systems). Here, we report the discovery of an MTG from Kutzneria albida (KalbTG), which exhibited no cross-reactivity with known MTG substrates or commonly used target proteins, such as antibodies. KalbTG was produced in Escherichia coli as soluble and active enzyme in the presence of its natural inhibitor ammonium to prevent potentially toxic cross-linking activity. The crystal structure of KalbTG revealed a conserved core similar to other MTGs but very short surface loops, making it the smallestMTGcharacterized to date. Ultra-dense peptide array technology involving a pool of 1.4 million unique peptides identified specific recognition motifs for KalbTG in these peptides. We determined that the motifs YRYRQ and RYESK are the best Gln and Lys substrates of KalbTG, respectively. By first reacting a bifunctionalized peptide with the more specific KalbTG and in a second step with the less specific MTG from Streptomyces mobaraensis, a successful bio-orthogonal labeling system was demonstrated. Fusing the KalbTG recognition motif to an antibody allowed for site-specific and ratio-controlled labeling using low label excess. Its site specificity, favorable kinetics, ease of use, and cost-effective production render KalbTG an attractive tool for a broad range of applications, including production of therapeutic antibody-drug conjugates. [ABSTRACT FROM AUTHOR]

Transglutaminases (TGs) play essential intracellular and extracellular roles by covalently cross-linking many proteins. Drosophila TG is encoded by one gene and has two alternative splicing-derived isoforms, TG-A and TG-B, which contain distinct N-terminal 46- and 38-amino acid sequences, respectively. The TGs identified to date do not have a typical endoplasmic reticulum (ER)-signal peptide, and the molecular mechanisms of their secretion under physiologic conditions are unclear. Immunocytochemistry revealed that TG-A localizes to multivesicular-like structures, whereas TG-B localizes to the cytosol. We also found that TG-A, but not TG-B, was modified concomitantly by N-myristoylation and S-palmitoylation, and N-myristoylation was a pre-requisite for S-palmitoylation. Moreover, TG-A, but not TG-B, was secreted in response to calcium signaling induced by Ca2+ ionophores and uracil, a pathogenic bacteria-derived substance. Brefeldin A and monensin, inhibitors of the ER/Golgi-mediated conventional pathway, did not suppress TG-A secretion, whereas inhibition of S-palmitoylation by 2-bromopalmitate blocked TG-A secretion. Ultracentrifugation, electron microscopy analyses, and treatments with inhibitors of multivesicular body formation revealed that TG-A was secreted via exosomes together with co-transfected mammalian CD63, an exosomal marker, and the secreted TG-A was taken up by other cells. The 8-residue N-terminal fragment of TG-A containing the fatty acylation sites was both necessary and sufficient for the exosome-dependent secretion of TG-A. In conclusion, TG-A is secreted through an unconventional ER/Golgi-independent pathway involving two types of fatty acylations and exosomes. [ABSTRACT FROM AUTHOR]

In Drosophila, the final immune deficiency (IMD) pathwaydependent signal is transmitted through proteolytic conversion of the nuclear factor-κB (NF-κB)-like transcription factor Relish to the active N-terminal fragment Relish-N. Relish-N is then translocated from the cytosol into the nucleus for the expression of IMD-controlled genes. We previously demonstrated that transglutaminase (TG) suppresses the IMD pathway by polymerizing Relish-N to inhibit its nuclear translocation. Conversely, we also demonstrated that orally ingested synthetic amines, such as monodansylcadaverine (DCA) and biotin-labeled pentylamine, are TG-dependently incorporated into Relish-N, causing the nuclear translocation of modified Relish-N in gut epithelial cells. It remains unclear, however, whether polyamine- containing Relish-N retains transcriptional activity. Here, we used mass spectrometry analysis of a recombinant Relish- N modified with DCA by TG activity after proteolytic digestion and show that the DCA-modified Gln residues are located in the DNA-binding region of Relish-N. TG-catalyzed DCA incorporation inhibited binding of Relish-N to the Rel-responsive element in the NF-κB-binding DNA sequence. Subcellular fractionation of TG-expressing Drosophila S2 cells indicated that TG was localized in both the cytosol and nucleus. Of note, natural polyamines, including spermidine and spermine, competitively inhibited TG-dependentDCAincorporation into Relish- N. Moreover, in vivo experiments demonstrated that Relish- N was modified by spermine and that this modification reduced transcription of IMD pathway-controlled cecropin A1 and diptericin genes. These findings suggest that intracellular TG regulates Relish-N-mediated transcriptional activity by incorporating polyamines into Relish-N and via protein-protein cross-linking. [ABSTRACT FROM AUTHOR]

Transglutaminase 2 (TG2) catalyzes transamidation or deamidation of its substrates and is ordinarily maintained in a catalytically inactive state in the intestine and other organs. Aberrant TG2 activity is thought to play a role in celiac disease, suggesting that a better understanding of TG2 regulation could help to elucidate the mechanistic basis of this malady. Structural and biochemical analysis has led to the hypothesis that extracellular TG2 activation involves reduction of an allosteric disulfide bond by thioredoxin-1 (TRX), but cellular and in vivo evidence for this proposal is lacking. To test the physiological relevance of this hypothesis, we first showed that macrophages exposed to pro-inflammatory stimuli released TRX in sufficient quantities to activate their extracellular pools of TG2. By using the C35S mutant of TRX, which formed a metastable mixed disulfide bond with TG2, we demonstrated that these proteins specifically recognized each other in the extracellular matrix of fibroblasts. When injected into mice and visualized with antibodies, we observed the C35S TRX mutant bound to endogenous TG2 as its principal protein partner in the small intestine. Control experiments showed no labeling of TG2 knock-out mice. Intravenous administration of recombinant TRX in wild-type mice, but not TG2 knock-out mice, led to a rapid rise in intestinal transglutaminase activity in a manner that could be inhibited by small molecules targeting TG2 or TRX. Our findings support the potential pathophysiological relevance of TRX in celiac disease and establish the Cys370-Cys371 disulfide bond of TG2 as one of clearest examples of an allosteric disulfide bond in mammals. [ABSTRACT FROM AUTHOR]

Transglutaminase 2 (TG2) is a Ca2+-dependent cross-linking enzyme involved in the pathogenesis of CD. We have previously characterized a panel of anti-TG2 mAbs generated from gut plasma cells of celiac patients and identified four epitopes (epitopes 1-4) located in the N-terminal part of TG2. Binding of the mAbs induced allosteric changes in TG2. Thus, we aimed to determine whether these mAbs could influence enzymatic activity through modulation of TG2 susceptibility to oxidative inactivation and Ca2+ affinity. All tested epitope 1 mAbs, as well as 679-14-D04, which recognizes a previously uncharacterized epitope, prevented oxidative inactivation and increased Ca2 sensitivity of TG2. We have identified crucial residues for binding of 679-14-D04 located within a Ca2+ binding site. Epitope 1 mAbs and 679-14-D04, although recognizing separate epitopes, behaved similarly when assessing their effect on TG2 conformation, suggesting that the shared effects on TG2 function can be explained by induction of the same conformational changes. None of the mAbs targeting other epitopes showed these effects, but epitope 2 mAbs reduced the rate of TG2-catalyzed reactions. Collectively, these effects could be relevant to the pathogenesis of CD. In A20 B cells transduced with TG2-specific B-cell receptor, epitope 2-expressing cells had poorer uptake of TG2-gluten complexes and were less efficient in gluten epitope presentation to T cells than cells expressing an epitope 1 receptor. Thus, the ability of epitope 1-targeting B cells to keep TG2 active and protected from oxidation might explain why generation of epitope 1-targeting plasma cells seems to be favored in celiac patients. [ABSTRACT FROM AUTHOR]

We recently reported that transglutaminase (TG) suppresses immune deficiency pathway-controlled antimicrobial peptides (IMD-AMPs), thereby conferring immune tolerance to gut microbes, and that RNAi of the TG gene in flies decreases the lifespan compared with non-TG-RNAi flies. Here, analysis of the bacterial composition of the Drosophila gut by next-generation sequencing revealed that gut microbiota comprising one dominant genus of Acetobacter in non-TG-RNAi flies was shifted to that comprising two dominant genera of Acetobacter and Providencia in TG-RNAi flies. Four bacterial strains, including Acetobacter persici SK1 and Acetobacter indonesiensis SK2, Lactobacillus pentosus SK3, and Providencia rettgeri SK4, were isolated from the midgut of TG-RNAi flies. SK1 exhibited the highest resistance to the IMD-AMPs Cecropin A1 and Diptericin among the isolated bacteria. In contrast, SK4 exhibited considerably lower resistance against Cecropin A1, whereas SK4 exhibited high resistance to hypochlorous acid. The resistance of strains SK1-4 against IMD-AMPs in in vitro assays could not explain the shift of the microbiota in the gut of TGRNAi flies. The lifespan was reduced in gnotobiotic flies that ingested both SK4 and SK1, concomitant with the production of reactive oxygen species and apoptosis in the midgut, whereas the survival rate was not altered in gnotobiotic flies that monoingested either SK4 or SK1. Interestingly, significant amounts of reactive oxygen species were detected in the midgut of gnotobiotic flies that ingested SK4 and SK2, concomitant with no significant apoptosis in the midgut. In gnotobiotic flies that co-ingested SK4 and SK1, an additional unknown factor(s) may be required to cause midgut apoptosis. [ABSTRACT FROM AUTHOR]

Transglutaminase from Streptomyces mobaraensis (MTG) is an important enzyme for cross-linking and modifying proteins. An intrinsic substrate of MTG is the dispase autolysis inducing protein (DAIP). The amino acid sequence of DAIP contains five potential glutamines and ten lysines for MTG mediated cross-linking. Aim of the study was to determine the structure and glutamine cross-linking sites of the first physiological MTG substrate. A production procedure was established in E. coli BL21 (DE3) to obtain high yields of recombinant DAIP. DAIP variants were prepared by replacing four out of five glutamines for asparagines in various combinations via site-directed mutagenesis. Incorporation of biotin cadaverine revealed preference of MTG for the DAIP glutamines in the order of Q39>>Q298>Q345~Q65>>Q144. In the structure of DAIP the preferred glutamines do cluster at the top of the seven-bladed β-propeller. This suggests a targeted cross-linking of DAIP by MTG that may occur after self-assembly in the bacterial cell wall. Based on our biochemical and the structural data of the first physiological MTG substrate, we further provide novel insight into determinants of MTG-mediated modification, specificity and efficiency. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) serve as a prime signal in the commitment to hematopoiesis in both mammals and Drosophila. In this study, the potential function of ROS during hematopoiesis in the crayfish Pacifastacus leniusculus was examined. The antioxidant N-acetylcysteine (NAC) was used to decrease ROSin both in vivo and in vitro experiments. Anincrease inROS was observed in the anterior proliferation center (APC) after LPS injection. In the absence of NAC, the LPS-induced increase in ROS levels resulted in the rapid restoration of the circulating hemocyte number. In the presence of NAC, a delay in the recovery rate of the hemocyte number was observed. NAC treatment also blocked the spread of APC and other hematopoietic tissue (HPT) cells, maintaining these cells at an undifferentiated stage. Extracellular transglutaminase (TGase) has been shown previously to play a role in maintaining HPT cells in an undifferentiated form. In this study, we show that extracellular TGase activity increased when the ROS level in HPT or APC cells was reduced after NAC treatment. In addition, collagen, a major component of the extracellular matrix and a TGase substrate were co-localized on the HPT cell surface. Taken together, the results of this study show that ROS are involved in crayfish hematopoiesis, in which a low ROS level is required to maintain hematopoietic progenitor cells in the tissue and to reduce hemocyte release. The potential roles of TGase in this process are investigated and discussed. [ABSTRACT FROM AUTHOR]

The main structural component of the mucus in the gastrointestinal tract is theMUC2mucin. It forms large networks that in colon build the loose outer mucous layer that provides the habitat for the commensal flora and the inner mucous layer that protects the epithelial cells by being impenetrable to bacteria. The epithelial cells in mice lacking MUC2 are not adequately protected from bacteria, resulting in inflammation and the development of colon cancer as found in human ulcerative colitis. Correct processing of the MUC2 mucin is the basis for the building of these protective networks. During the biosynthesis of the MUC2 mucin, post-translational modifications are formed resulting in reduction-insensitive bonds betweenMUC2 monomers. By the use of γ-glutamyltranspeptidase and isopeptidase activity in leech saliva, we could show that the molecular nature of these reduction-insensitive bonds is isopeptide bonds formed between side chains of lysine and glutamine. Transglutaminase 2 has an affinity to the MUC2 CysD2 domain in the nanomolar range and can catalyze its cross-linking. By using mass spectrometry, we identified MUC2 residues involved in this cross-linking. This shows for the first time that transamidation is not only stabilizing the skin and the fibrin clot, but is also important for the correct intracellular processing of MUC2 to generate protective mucus. [ABSTRACT FROM AUTHOR]

Tissue transglutaminase (tTG) is an acyltransferase/GTPbinding protein that contributes to the development of various diseases. In human cancer cells, tTG activates signaling pathways that promote cell growth and survival, whereas in other disorders (i.e. neurodegeneration), overexpression of tTG enhances cell death. Therefore, it is important to understand how tTG is differentially regulated and functioning to promote diametrically distinct cellular outcomes. Previous structural studies revealed that tTG adopts either a nucleotide-bound closed conformation or a transamidation-competent open conformation. Here we provide evidence showing that these different conformational states determine whether tTG promotes, or is detrimental to, cell survival, with the open conformation of the protein being responsible for inducing cell death. First, we demonstrate that a nucleotide binding-defective form of tTG, which has previously been shown to induce cell death, assumes an open conformation in solution as assessed by an enhanced sensitivity to trypsin digestion and by small angle x-ray scattering (SAXS) analysis. We next identify two pairs of intramolecular hydrogen bonds that, based on existing x-ray structures, are predicted to form between the most C-terminal β-barrel domain and the catalytic core domain of tTG. By disrupting these hydrogen bonds, we are able to generate forms of tTG that constitutively assume an open conformation and induce apoptosis. These findings provide important insights into how tTG participates in the pathogenesis of neurodegenerative diseases, particularly with regard to the actions of a C-terminal truncated form of tTG (TG-Short) that has been linked to such disorders and induces apoptosis by assuming an open-like conformation. [ABSTRACT FROM AUTHOR]

Antibodies to the autoantigen transglutaminase 2 (TG2) are a hallmark of celiac disease. We have studied the interaction between TG2 and an anti-TG2 antibody (679-14-E06) derived from a single gut IgA plasma cell of a celiac disease patient. The antibody recognizes one of four identified epitopes targeted by antibodies of plasma cells of the disease lesion. The binding interface was identified by small angle X-ray scattering, ab initio and rigid body modeling using the known crystal structure of TG2 and the crystal structure of the antibody Fab fragment, which was solved at 2.4 Å resolution. The result was confirmed by testing binding of the antibody to TG2 mutants by ELISA and surface plasmon resonance. TG2 residues R116 and H134 were identified to be critical for binding of 679-14-E06 as well as other epitope 1 antibodies. In contrast, antibodies directed toward the two other main epitopes (epitopes 2 and 3) were not affected by these mutations. Molecular dynamics simulations suggest interactions of 679-14-E06 with the N-terminal domain of TG2 via the CDR2 and CDR3 loops of the heavy chain and the CDR2 loop of the light chain. In addition there were contacts of the framework 3 region of the heavy chain with the catalytic domain of TG2. The results provide an explanation for the biased usage of certain heavy and light chain gene segments by epitope 1-specific antibodies in celiac disease. [ABSTRACT FROM AUTHOR]

Streptococcus suis serotype 2 (Ss2) is an important swine and human zoonotic pathogen. In the present study, we identified a novel secreted immunogenic protein, SsTGase, contained a highly conserved eukaryotic-like transglutaminase (TGase) domain at the N terminus. We found that inactivation of SsTGase significantly reduced the virulence of Ss2 in a pig infection model and impaired its anti-phagocytosis in human blood. We further solved the crystal structure of the N-terminal portion of the protein in homodimer form at 2.1 Å. Structure based mutagenesis and biochemical studies suggested that disruption of the homodimer directly resulted in the loss of its TGase activity and anti-phagocytic ability. Characterization of SsTGase as a novel virulence factor of Ss2 by acting as a TGase would be beneficial for developing new therapeutic agents against Ss2 infection. [ABSTRACT FROM AUTHOR]

The molecular mechanism of transglutaminase II (TGaseII)-mediated allergic inflammation remains largely unknown. TGaseII, induced by antigen stimulation, showed an interaction and co-localization with FcεRI. TGaseII was necessary for in vivo allergic inflammation, such as triphasic cutaneous reaction, passive cutaneous anaphylaxis, and passive systemic anaphylaxis. TGaseII was necessary for the enhanced metastatic potential of B16F1 melanoma cells by passive systemic anaphylaxis. TGaseII was shown to be a secreted protein. Recombinant TGaseII protein increased the histamine release and β-hexosaminidase activity, and enhanced the metastatic potential of B16F1 mouse melanoma cells. Recombinant TGaseII protein induced the activation of EGF receptor and an interaction betweenEGFreceptor and FcεRI. Recombinant TGaseII protein displayed angiogenic potential accompanied by allergic inflammation. R2 peptide, an inhibitor of TGaseII, exerted negative effects on in vitro and in vivo allergic inflammationbyregulating the expression of TGaseII and FcεRI signaling. MicroRNA (miR)-218 and miR-181a, decreased during allergic inflammation, were predicted as negative regulators of TGaseII by microRNA array and TargetScan analysis. miR-218 and miR-181a formed a negative feedback loop with TGaseII and regulated the in vitro and in vivo allergic inflammation. TGaseII was necessary for the interaction between mast cells and macrophages during allergic inflammation. Mast cells and macrophages, activated during allergic inflammation, were responsible for the enhanced metastatic potential of tumor cells that are accompanied by allergic inflammation. In conclusion, the TGaseII/miR-218/-181a feedback loop can be employed for the development of anti-allergy therapeutics. [ABSTRACT FROM AUTHOR]

Background: Tissue transglutaminase (tTG) promotes various aspects of oncogenesis, including cell survival. Results: Ectopically expressed tTG in non-transformed cells triggers a survival response that involves c-Src and PI3-kinase. Conclusion: tTG promotes survival by activating PI3-kinase through a c-Src-dependent mechanism. Significance: These findings demonstrate that tTG has an intrinsic capability to promote cell survival and explains how it contributes to oncogenesis. Tissue transglutaminase (tTG) functions as a GTPase and an acyl transferase that catalyzes the formation of protein crosslinks. tTG expression is frequently up-regulated in human cancer, where it has been implicated in various aspects of cancer progression, including cell survival and chemo-resistance. However, the extent to which tTG cooperates with other proteins within the context of a cancer cell, versus its intrinsic ability to confer transformed characteristics to cells, is poorly understood. To address this question, we asked what effect the ectopic expression of tTG in a non-transformed cellular background would have on the behavior of the cells. Using NIH3T3 fibroblasts stably expressing a Myc-tagged form of tTG, we found that tTG strongly protected these cells from serum starvationinduced apoptosis and triggered the activation of the PI3-kinase/ mTOR Complex 1 (mTORC1)/p70 S6-kinase pathway. We determined that tTG forms a complex with the non-receptor tyrosine kinase c-Src and PI3-kinase, and that treating cells with inhibitors to block tTG function (monodansylcadaverine; MDC) or c-Src kinase activity (PP2) disrupted the formation of this complex, and prevented tTG from activating the PI3-kinase pathway. Moreover, treatment of fibroblasts over-expressing tTG with PP2, or with inhibitors that inactivate components of the PI3-kinase pathway, including PI3-kinase (LY294002) and mTORC1 (rapamycin), ablated the tTG-promoted survival of the cells. These findings demonstrate that tTG has an intrinsic capability to stimulate cell survival through a novel mechanism that activates PI3-kinase signaling events, thus highlighting tTG as a potential target for the treatment of human cancer. [ABSTRACT FROM AUTHOR]

Coagulation factor XIII (FXIII) is a transglutaminase with a well defined role in the final stages of blood coagulation. Active FXIII (FXIIIa) catalyzes the formation of ∈-(γ-glutamyl)lysine isopeptide bonds between specific Gln and Lys residues. The primary physiological outcome of this catalytic activity is stabilization of the fibrin clot during coagulation. The stabilization is achieved through the introduction of cross-links between fibrin monomers and through cross-linking of proteins with anti-fibrinolytic activity to fibrin. FXIIIa additionally cross-links several proteins with other functionalities to the clot. Cross-linking of proteins to the clot is generally believed to modify clot characteristics such as proteolytic susceptibility and hereby affect the outcome of tissue damage. In the present study, we use a proteomic approach in combination with transglutaminase-specific labeling to identify FXIIIa plasma protein substrates and their reactive residues. The results revealed a total of 147 FXIIIa substrates, of which 132 have not previously been described. We confirm that 48 of the FXIIIa substrates were indeed incorporated into the insoluble fibrin clot during the coagulation of plasma. The identified substrates are involved in, among other activities, complement activation, coagulation, inflammatory and immune responses, and extracellular matrix organization. [ABSTRACT FROM AUTHOR]

Corneal stroma contains an extracellular matrix of orthogonal lamellae formed by parallel and equidistant fibrils with a homogeneous diameter of ~35 nm. This is indispensable for corneal transparency and mechanical functions. However, the mechanisms controlling corneal fibrillogenesis are incompletely understood and the conditions required for lamellar stacking are essentially unknown. Under appropriate conditions, chick embryo corneal fibroblasts can produce an extracellular matrix in vitro resembling primary corneal stroma during embryonic development. Among other requirements, cross-links between fibrillar collagens, introduced by tissue transglutaminase-2, are necessary for the self-assembly of uniform, small diameter fibrils but not their lamellar stacking. By contrast, the subsequent lamellar organization into plywood-like stacks depends on lysyl aldehyde-derived cross-links introduced by lysyl oxidase activity, which, in turn, only weakly influences fibril diameters. These cross-links are introduced at early stages of fibrillogenesis. The enzymes are likely to be important for a correct matrix deposition also during repair of the cornea. [ABSTRACT FROM AUTHOR]

Pituitary adenylate cyclase-activating polypeptide (PACAP) functions as a neuroprotective factor through the PACAP type 1 receptor, PAC1. In a previous work, we demonstrated that nerve growth factor augmented PAC1 gene expression through the activation of Sp1 via the Ras/MAPK pathway. We also observed that PAC1 expression in Neuro2a cells was transiently suppressed during in vitro ischemic conditions, oxygen-glucose deprivation (OGD). Because endoplasmic reticulum (ER) stress is induced by ischemia, we attempted to clarify how ER stress affects the expression of PAC1. Tunicamycin, which induces ER stress, significantly suppressed PAC1 gene expression, and salubrinal, a selective inhibitor of the protein kinase RNA-like endoplasmic reticulum kinase signaling pathway of ER stress, blocked the suppression. In luciferase reporter assay, we found that two Sp1 sites were involved in suppression of PAC1 gene expression due to tunicamycin or OGD. Immunocytochemical staining demonstrated that OGD-induced transglutaminase 2 (TG2) expression was suppressed by salubrinal or cystamine, a TG activity inhibitor. Further, the OGD-induced accumulation of cross-linked Sp1 in nuclei was suppressed by cystamine or salubrinal. Together with cystamine, R283, TG2-specific inhibitor, and siRNA specific for TG2 also ameliorated OGD-induced attenuation of PAC1 gene expression. These results suggest that Sp1 cross-linking might be crucial in negative regulation of PAC1 gene expression due to TG2 in OGD-induced ER stress. [ABSTRACT FROM AUTHOR]

Mutations in matrix Gla protein (MGP) have been correlated with vascular calcification. In the mouse model, MGP null vascular disease presents as calcifying cartilaginous lesions and mineral deposition along elastin lamellae (elastocalcinosis). Here we examined the mechanisms underlying both of these manifestations. Genetic ablation of enzyme transglutaminase 2 (TG2) in Mgp-/- mice dramatically reduced the size of cartilaginous lesions in the aortic media, attenuated calcium accrual more than 2-fold, and doubled longevity as compared with control Mgp-/- animals. Nonetheless, the Mgp-/-;Tgm2-/- mice still died prematurely as compared with wild-type and retained the elastocalcinosis phenotype. This pathology in Mgp-/- animals was developmentally preceded by extensive fragmentation of elastic lamellae and associated with elevated serine elastase activity in aortic tissue and vascular smooth muscle cells. Systematic gene expression analysis followed by an immunoprecipitation study identified adipsin as the major elastase that is induced in the Mgp-/- vascular smooth muscle even in the TG2 null background. These results reveal a central role for TG2 in chondrogenic transformation of vascular smooth muscle and implicate adipsin in elastin fragmentation and ensuing elastocalcinosis. The importance of elastin calcification in MGP null vascular disease is highlighted by significant residual vascular calcification and mortality in Mgp-/-;Tgm2-/- mice with reduced cartilaginous lesions. Our studies identify two potential therapeutic targets in vascular calcification associated with MGP dysfunction and emphasize the need for a comprehensive approach to this multifaceted disorder. [ABSTRACT FROM AUTHOR]

Anopheline mosquitoes are the only vectors of human malaria worldwide. It is now widely accepted that mosquito immune responses play a crucial role in restricting Plasmodium development within the vector; therefore, further dissection of the molecular mechanisms underlying these processes should inform new vector control strategies urgently needed to roll back the disease. Here, using genome-wide transcriptional profiling, bioinformatics, and functional gene analysis, we identify a new axis of mosquito resistance to monoclonalPlasmodium falciparum infections that includes the AP-1 transcription factor Fos and the transglutaminase 2 (TGase2), a cross-linking enzyme with known roles in wound responses. We demonstrate that Fos regulates induction of TGase2expression after wounding but does not affect expression of the components of the well characterized complement-like system. Silencing of Fos or of TGase2aborts the wounding-induced mosquito killing of P. falciparum. These results reveal multiple signaling pathways that are required for efficient Plasmodiumkilling in Anopheles gambiae. [ABSTRACT FROM AUTHOR]

Male Anopheles mosquitoes coagulate their seminal fluids via cross-linking of a substrate, called Plugin, by the seminal transglutaminase AgTG3. Formation of the "mating plug" by crosslinking Plugin is necessary for efficient sperm storage by females. AgTG3 has a similar degree of sequence identity (∼30%) to both human Factor XIII (FXIII) and tissue transglutaminase 2 (hTG2). Here we report the solution structure and in vitro activity for the cross-linking reaction of AgTG3 and Plugin. AgTG3 is a dimer in solution and exhibits Ca2+-dependent nonproteolytic activation analogous to cytoplasmic FXIII. The C-terminal domain of Plugin is predominantly α-helical with extended tertiary structure and oligomerizes in solution. The specific activity of AgTG3 was measured as 4.25 × 10-2 units mg-1. AgTG3 is less active than hTG2 assayed using the general substrateTVQQELbut has 8-10×higher relative activity when Plugin is the substrate. Mass spectrometric analysis of cross-linked Plugin detects specific peptides including a predicted consensus motif for cross-linking by AgTG3. These results support the development of AgTG3 inhibitors as specific and effective chemosterilants for A. gambiae. [ABSTRACT FROM AUTHOR]

Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme that cross-links proteins and its overexpression, linked to a drug resistant phenotype, is commonly observed in cancer cells. Further, up-regulation ofTG2expression occurs during response to various forms of cell stress; however, the molecular mechanisms that drive inducible expression of the TG2 gene (TGM2) require elucidation. Here we show that genotoxic stress induces TG2 expression through the Ataxia-Telangiectasia, Mutated (ATM)/ Nuclear Factor κ light chain enhancer of activated B cells (NFκB) signaling pathway. We further document that NFκB is both necessary and sufficient to drive constitutive TG2 expression in cultured cell lines. Additionally, shRNA-mediated knockdown or pharmacological inhibition of the ATM kinase results in reduced constitutive TG2 expression and NFκB transcriptional activity. We document that the NFκB subunit p65 (RelA) interacts with two independent consensus NFκB binding sites within the TGM2 promoter, that mutation of either site or pharmacological inhibition of NFκB reduces TGM2 promoter activity, and genotoxic stress drives heightened association of p65 with the TGM2 promoter. Finally, we observed that knockdown of either p65 orATMin MDA-MB-468 breast cancer cells expressing recombinant TG2 partially reduces resistance to doxorubicin, indicating that the drug resistance linked to overexpression of TG2 functions, in part, through p65 and ATM. This work establishes a novel ATM-dependent signaling loop where TG2 and NFκB activate each other resulting in sustained activation of NFκB and acquisition of a drug-resistant phenotype. [ABSTRACT FROM AUTHOR]

Heparan sulfate proteoglycans are critical binding partners for extracellular tranglutaminase-2 (TG2), a multifunctional protein involved in tissue remodeling events related to organ fibrosis and cancer progression. We previously showed that TG2 has a strong affinity for heparan sulfate (HS)/heparin and reported that the heparan sulfate proteoglycan syndecan-4 acts as a receptor for TG2 via its HS chains in two ways: by increasing TG2-cell surface trafficking/externalization and by mediating RGD-independent cell adhesion to fibronectin-TG2 matrix during wound healing. Here we have investigated the molecular basis of this interaction. Site-directed mutagenesis revealed that either mutation of basic RRWK (262-265) or KQKRK (598-602) clusters, forming accessible heparin binding sequences on the TG2 three-dimensional structure, led to an almost complete reduction of heparin binding, indicating that both clusters contribute to form a single binding surface. Mutation of residues Arg19 and Arg28 also led to a significant reduction in heparin binding, suggesting their involvement. Our findings indicate that the heparin binding sites on TG2 mainly comprise two clusters of basic amino acids, which are distant in the linear sequence but brought into spatial proximity in the folded "closed" protein, forming a high affinity heparin binding site. Molecular modeling showed that the identified site can make contact with a single heparin-derived pentasaccharide. The TG2-heparin binding mutants supported only weak RGD-independent cell adhesion compared with wild type TG2 or mutants with retained heparin binding, and both heparin binding clusters were critical for TG2-mediated cell adhesion. These findings significantly advance our knowledge of how HS/heparin influences the adhesive function of TG2. [ABSTRACT FROM AUTHOR]

Transglutaminase 2 (TG2) is a versatile protein that isimplicated in significant biological processes, including cell death and degenerative diseases. A possible role of TG2 in the apoptotic death of cancer cells induced by photodynamic therapy (PDT) was suggested recently; however, the mechanism by which TG2 regulates apoptotic responses to PDT remains to be elucidated. In this study, we investigated the key signaling pathways stimulated during apoptotic cell death following PDT and whether inhibition of TG2 activation using pharmacological approaches and siRNAs affects the signaling pathways. PDT caused the release of both cytochrome c and apoptosis-inducing factor (AIF) by damaging mitochondria, which resulted in caspase-dependent and caspase-independent apoptotic cell death, respectively. Released AIF translocated to the nucleus and, synergistically with the caspase-dependent pathway, led to apoptotic cell death. Both the caspase cascade and the activation of AIF following PDT were mediated by TG2 activation. In addition, PDT-activated calpain was responsible for the sequential events of Bax translocation, the collapse ofΔψm' caspase-3 activation, and AIF translocation, all of which were provoked by TG2 activation. Together, these results demonstrate that PDT with a chlorin-based photosensitizer targets TG2 by activating calpaininduced Bax translocation, which induces apoptotic cell death through both caspase-dependent and AIF-mediated pathways. Moreover, these results indicate that TG2 may be a possible therapeutic target for PDT treatment of cancer. [ABSTRACT FROM AUTHOR]

Tissue transglutaminase (TG2) is a multifunctional Ca2+-activated protein cross-linking enzyme secreted into the extracellular matrix (ECM), where it is involved in wound healing and scarring, tissue fibrosis, celiac disease, and metastatic cancer. Extracellular TG2 can also facilitate cell adhesion important in wound healing through a nontransamidating mechanism via its association with fibronectin, heparan sulfates (HS), and integrins. Regulating the mechanism how TG2 is translocated into the ECM therefore provides a strategy for modulating these physiological and pathological functions of the enzyme. Here, through molecular modeling and mutagenesis, we have identified the HS-binding site of TG2 202KFLKNAGRDCSRRSSPVYVGR222. We demonstrate the requirement of this binding site for translocation of TG2 into the ECM through a mechanism involving cell surface shedding of HS. By synthesizing a peptide NPKFLKNAGRDCSRRSS corresponding to the HS-binding site within TG2, we also demonstrate how this mimicking peptide can in isolation compensate for the RGD-induced loss of cell adhesion on fibronectin via binding to syndecan-4, leading to activation of PKCα, pFAK-397, and ERK1/2 and the subsequent formation of focal adhesions and actin cytoskeleton organization. A novel regulatory mechanism for TG2 translocation into the extracellular compartment that depends upon TG2 conformation and the binding of HS is proposed. [ABSTRACT FROM AUTHOR]

Transglutaminases (TGases) are ubiquitous enzymes that catalyze selective cross-linking between protein-bound glutamine and lysine residues; the resulting isopeptide bond confers high resistance to proteolysis. Phytophthora sojae, a pathogen of soybean, secretes a Ca2+-dependent TGase (GP42) that is activating defense responses in both host and non-host plants. A GP42 fragment of 13 amino acids, termed Pep-13, was shown to be absolutely indispensable for both TGase and elicitor activity. GP42 does not share significant primary sequence similarity with known TGases from mammals or bacteria. This suggests that GP42 has evolved novel structural and catalytic features to support enzymatic activity. We have solved the crystal structure of the catalytically inactive point mutant GP42 (C290S) at 2.95 Å resolution and identified residues involved in catalysis by mutational analysis. The protein comprises three domains that assemble into an elongated structure. Although GP42 has no structural homolog, its core region displays significant similarity to the catalytic core of the Mac-1 cysteine protease from Group A Streptococcus, a member of the papain-like superfamily of cysteine proteases. Proteins that are taxonomically related to GP42 are only present in plant pathogenic oomycetes belonging to the order of the Peronosporales (e.g. Phytophthora, Hyaloperonospora, and Pythium spp.) and in marine Vibrio bacteria. This suggests that a lateral gene transfer event may have occurred between bacteria and oomycetes. Our results offer a basis to design and use highly specific inhibitors of the GP42-like TGase family that may impair the growth of important oomycete and bacterial pathogens. [ABSTRACT FROM AUTHOR]

Protein glutaminase, which converts a protein glutamine residue to a glutamate residue, is expected to be useful as a new food-processing enzyme. The crystal structures of the mature and pro forms of the enzyme were refined at 1.15 and 1.73 Å resolution, respectively. The overall structure of the mature enzyme has a weak homology to the core domain of human transglutaminase-2. The catalytic triad (Cys-His-Asp) common to transglutaminases and cysteine proteases is located in the bottom of the active site pocket. The structure of the recombinant pro form shows that a short loop between S2 and S3 in the proregion covers and interacts with the active site of the mature region, mimicking the protein substrate of the enzyme. Ala-47 is located just above the pocket of the active site. Two mutant structures (A47Q-1 and A47Q-2) refined at 1.5 Å resolution were found to correspond to the enzyme-substrate complex and an S-acyl intermediate. Based on these structures, the catalytic mechanism of protein glutaminase is proposed. [ABSTRACT FROM AUTHOR]

Cell migration is essential for several important biological outcomes and is involved in various developmental disorders and disease states including cancer cell invasiveness and metastasis. A fundamental step in cell migration is the development of a leading edge. By using HeLa carcinoma cells as an initial model system, we uncovered a surprising role for the heat shock protein 70 (Hsp70) and its ability to bind the protein cross-linking enzyme, tissue transglutaminase (tTG), in cancer cell migration. Treatment of HeLa cells with EGF results in the activation of a plasma membrane-associated pool of tTG and its redistribution to the leading edges of these cells, which are essential events for EGF-stimulated HeLa cell migration. However, we then found that the ability of tTG to be localized to the leading edge is dependent on Hsp70. Similarly, the localization of tTG to the leading edges of MDAMB231 breast carcinoma cells, where it also plays an essential role in their migration, has a strict requirement for Hsp70. Treatment of these different cell lines with inhibitors against the ATP hydrolytic activity of Hsp70 prevented tTG from localizing to their leading edges and thereby blocked EGF-stimulated HeLa cell migration, as well as the constitutive migration normally exhibited by MDAMB231 cells. These findings highlight a new and unconventional role for the chaperonin activity of Hsp70 in the localization of a key regulatory protein (tTG) at the leading edges of cancer cells and the important consequences that this holds for their ability to migrate. [ABSTRACT FROM AUTHOR]

Transglutaminase 2 (TG2) is secreted by a non-classical pathway into the extracellular space, where it has several activities pertinent to fibronectin (FN), including binding to the gelatin-binding domain of FN and acting as an integrin co-receptor. Glutamines in the N-terminal tail of FN are known to be susceptible to transamidation by both TG2 and activated blood coagulation factor XIII (FXIIIa). We used immunoblotting, limited proteolysis, and mass spectrometry to localize glutamines within FN that are subject to TG2-catalyzed incorporation of dansylcadaverine in comparison to residues modified by FXIIIa. Such analysis of plasma FN indicated that Gln-3, Gln-7, and Gln-9 in the N-terminal tail and Gln-246 of the linker between fifth and sixth type I modules (5F1 and 6F1) are transamidated by both enzymes. Only minor incorporation of dansylcadaverine was detected elsewhere. Labeling of C-terminally truncated FN constructs revealed efficient TG2- or FXIIIa-catalyzed dansylcadaverine incorporation into the N-terminal residues of constructs as small as the 29-kDa fragment that includes 1-5F1 and lacks modules from the adjacent gelatin-binding domain. However, when only 1-3F1 were present, dansylcadaverine incorporation into the N-terminal residues of FN was lost and instead was in the enzymes, near the active site of TG2 and terminal domains of FXIIIa. Thus, these results demonstrate that FXIIIa and TG2 act similarly on glutamines at either end of 1-5F1 and transamidation specificity of both enzymes is achieved through interactions with the intact 29K fragment. [ABSTRACT FROM AUTHOR]

Transglutaminase type 2 (TG2) catalyzes the formation of an ϵ-(γ-glutamyl)-lysine isopeptide bond between adjacent peptides or proteins including those of the extracellular matrix (ECM). Elevated extracellular TG2 leads to accelerated ECM deposition and reduced clearance that underlie tissue scarring and fibrosis. The extracellular trafficking of TG2 is crucial to its role in ECM homeostasis; however, the mechanism by which TG2 escapes the cell is unknown as it has no signal leader peptide and therefore cannot be transported classically. Understanding TG2 transport may highlight novel mechanisms to interfere with the extracellular function of TG2 as isoform-specific TG2 inhibitors remain elusive. Mammalian expression vectors were constructed containing domain deletions of TG2. These were transfected into three kidney tubular epithelial cell lines, and TG2 export was assessed to identify critical domains. Point mutation was then used to highlight specific sequences within the domain required for TG2 export. The removal of β-sandwich domain prevented all TG2 export. Mutations of Asp94 and Asp97 within the N-terminal β-sandwich domain were identified as crucial for TG2 externalization. These form part of a previously identified fibronectin binding domain (88WTATVVDQQDCTLSLQLTT106). However, siRNA knockdown of fibronectin failed to affect TG2 export. The sequence 88WTATVVDQQDCTLSLQLTT106 within the β-sandwich domain of TG2 is critical to its export in tubular epithelial cell lines. The extracellular trafficking of TG2 is independent of fibronectin. [ABSTRACT FROM AUTHOR]

Acquired chemoresistance not only blunts anticancer therapy but may also promote cancer cell migration and metastasis. Our previous studies have revealed that acquired tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance in lung cancer cells is associated with Akt-mediated stabilization of cellular caspase 8 and Fas-associated death domain (FADD)-like apoptosis regulator-like inhibitory protein (c-FLIP) and myeloid cell leukemia 1 (Mcl-1). In this report, we show that cells with acquired TRAIL resistance have significantly increased capacities in migration and invasion. By gene expression screening, tissue transglutaminase (TGM2) was identified as one of the genes with the highest expression increase in TRAIL-resistant cells. Suppressing TGM2 dramatically alleviated TRAIL resistance and cell migration, suggesting that TGM2 contributes to these two phenotypes in TRAIL-resistant cells. TGM2-mediated TRAIL resistance is likely through c-FLIP because TGM2 suppression significantly reduced c-FLIP but not Mcl-1 expression. The expression of matrix metalloproteinase 9 (MMP-9) was suppressed when TGM2 was inhibited, suggesting that TGM2 potentiates cell migration through up-regulating MMP-9 expression. We found that EGF receptor (EGFR) was highly active in the TRAIL-resistant cells, and suppression of EGFR dramatically reduced TGM2 expression. We further determined JNK and ERK, but not Akt and NF-κB, are responsible for EGFR-mediated TGM2 expression. These results identify a novel pathway that involves EGFR, MAPK (JNK and ERK), and TGM2 for acquired TRAIL resistance and cell migration in lung cancer cells. Because TGM2 couples TRAIL resistance and cell migration, it could be a molecular target for circumventing acquired chemoresistance and metastasis in lung cancer. [ABSTRACT FROM AUTHOR]

Aberrant pyroglutamate formation at the N terminus of certain peptides and proteins, catalyzed by glutaminyl cyclases (QCs), is linked to some pathological conditions, such as Alzheimer disease. Recently, a glutaminyl cyclase (QC) inhibitor, PBD 150, was shown to be able to reduce the deposition of pyrogluta mate-modified amyloid-peptides in brain of transgenic mouse models of Alzheimer disease, leading to a significant improvement of learning and memory in those transgenic animals. Here, we report the 1.05-1.40. Å resolution structures, solved by the sulfur single-wavelength anomalous dispersion phasing method, of the Golgi-luminal catalytic domain of the recently identified Golgi-resident QC (gQC) and its complex with PBD150. We also describe the high-resolution structures of secretory QC (sQC)-PBD150 complex and two other gQC- inhibitor complexes. gQC structure has a scaffold similar to that of sQC but with a relatively wider and negatively charged active site, sggesting a distinct substrate specificity from sQC. Upon binding to PBD15O, a large loop movement in gQC allows the inhibitor to be tightly held in its active site primarily by hydrophobic interactions. Further comparisons of the inhibitor-bound structures revealed distinct interactions of the inhibitors with gQC and sQC, which are consistent with the results from our inhibitor assays reported here. Because gQC and sQC may play different biological roles in vivo, the different inhibitor binding modes allow the design of specific inhibitors toward gQC and sQC. [ABSTRACT FROM AUTHOR]

Tissue transglutaminase (TGase) has been implicated in a number of cellular processes and disease states, where the enzymatic actions of TGase may serve in both, cell survival and apoptosis. To date, the precise functional properties of TGase in cell survival or cell death mechanisms still remain elusive. TGase- mediated cross-linking has been reported to account for the formation of insoluble lesions in conformational diseases. We report here that TGase induces intramolecular cross-linking of β-amyloid peptide (An), resulting in structural changes of monomeric Aβ. Using high resolution mass spectrometry (MS) of cross-linked Aβpeptides, we observed a shift in mass, which is, presumably associated with the loss of NH3 due to enzymatic transamidation activity and hence intramolecular peptide cross-linking. We have observed that a large population of Aβ monomers contained an 0,984 Da increase in mass at a glutamine residue, indicating that glutamine 15 serves as an indispensable substrate in TGase-mediated deamidation to glutamate 15. We provide strong analytical evidence on TGase- mediated Aβ peptide dimerization, through covalent intermolecular cross-linking and hence the formation of Aβ1-40 dimers. Our in depth analyses indicate that TGase-induced post-translational modifications of Aβ peptide may serve as an important seed for aggregation. [ABSTRACT FROM AUTHOR]

Factor XIII (FXIII) is a pro-transglutaminase found in the plasma as well as intracellularly in platelets and macrophages. Plasma FXIII is activated by thrombin cleavage (FXIIIa*) and acts in the final stages of blood coagulation cascade. In contrast, the function and activation of cellular FXIII are less characterized. Cellular FXIII relies on a conformational activation of the protein. The nonproteolytic activation of FXIII to FXIIIå induced by Ca2+ alone is well known, but up until now it has been discussed under which conditions the process can be induced and whether it can be reversed. Here, we study the nature of the Ca2+-induced FXIII activation. Previously used methods to evaluate FXIII activity detect both FXIIIa* and FXIIIå because they rely on occurrence of enzyme activity or on active site Cys-314 solvent accessibility. Therefore, an analytical HPLC method was developed that separates zymogen recombinant FXIII (rFXIII) from rFXIIIå. The data demonstrate that nonproteolytic activation and deactivation are highly dependent on Ca2+ concentration, buffer, and salt components. Moreover, it is established that Ca2+ activation of rFXIII is fully reversible, and only 2-5 mm CaCl2 is sufficient to retain full rFXIIIå activity. However, below 2 mm CaCl2 the rFXIIIå molecule deactivates. The deactivated molecule can subsequently undergo a new activation round. Furthermore, it is demonstrated that thermal stress of freeze-dried rFXIII can induce a new predisposed form that activates faster than nonstressed rFXIII. [ABSTRACT FROM AUTHOR]

The crystal structure of the microbial transglutaminase (MTGase) zymogen from Streptomyces mobaraense has been determined at 1.9-Å resolution using the molecular replacement method based on the crystal structure of the mature MTGase. The overall structure of this zymogen is similar to that of the mature form, consisting of a single disk-like domain with a deep active cleft at the edge of the molecule. A major portion of the prosequence (45 additional amino acid residues at the N terminus of the mature transglutaminase) folds into an L-shaped structure, consisting of an extended N-terminal segment linked with a one-turn short helix and a long α-helix. Two key residues in the short helix of the prosequence, Tyr-12 and Tyr-16, are located on top of the catalytic triad (Cys-110, Asp-301, and His-320) to block access of the substrate acyl donors and acceptors. Biochemical characterization of the mature MTGase, using N-α-benzyloxycarbonyl-l-glutaminylglycine as a substrate, revealed apparent Km and kcat/Km values of 52.66 mm and 40.42 mm-1 min-1, respectively. Inhibition studies using the partial prosequence SYAETYR and homologous sequence SQAETYR showed a noncompetitive inhibition mechanism with IC50 values of 0.75 and 0.65 mm, respectively, but no cross-linking product formation. Nevertheless, the prosequence homologous oligopeptide SQAETQR, with Tyr-12 and Tyr-16 each replaced with Gln, exhibited inhibitory activity with the formation of the SQAETQR-monodansylcadaverine fluorophore cross-linking product (SQAETQR-C-DNS). MALDI-TOF tandem MS analysis of SQAETQR-C-DNS revealed molecular masses corresponding to those of NSQAETQC-C-DNS and C-DNS-NQRC sequences, suggesting the incorporation of C-DNS onto the C-terminal Gln residue of the prosequence homologous oligopeptide. These results support the putative functional roles of both Tyr residues in substrate binding and inhibition. [ABSTRACT FROM AUTHOR]

Although both metastatic tumor antigen 1 (MTA1), a master chromatin modifier, and transglutaminase 2 (TG2), a multifunctional enzyme, are known to be activated during inflammation, it remains unknown whether these molecules regulate inflammatory response in a coordinated manner. Here we investigated the role of MTA1 in the regulation of TG2 expression in bacterial lipopolysaccharide (LPS)-stimulated mammalian cells. While studying the impact of MTA1 status on global gene expression, we unexpectedly discovered that MTA1 depletion impairs the basal as well as the LPS-induced expression of TG2 in multiple experimental systems. We found that TG2 is a chromatin target of MTA1 and of NF-κB signaling in LPS-stimulated cells. In addition, LPS-mediated stimulation of TG2 expression is accompanied by the enhanced recruitment of MTA1, p65RelA, and RNA polymerase II to the NF-κB consensus sites in the TG2 promoter. Interestingly, both the recruitment of p65 and TG2 expression are effectively blocked by a pharmacological inhibitor of the NF-κB pathway. These findings reveal an obligatory coregulatory role of MTA1 in the regulation of TG2 expression and of the MTA1-TG2 pathway, at least in part, in LPS modulation of the NF-κB signaling in stimulated macrophages. [ABSTRACT FROM AUTHOR]

Transglutaminase 2 (TG2) in the extracellular matrix is largely inactive but is transiently activated upon certain types inflammation and cell injury. The enzymatic activity of extracelular TG2 thus appears to be tightly regulated. As TG2 is know to be sensitive to changes in the redox environment, inactivation through oxidation presents a plausible mechanism. Using mass spectrometry, we have identified a redox-sensitive cysteine triad consisting of Cys230, Cys370 and Cys371 that is involved in oxidative inactivation of TG2. Within this triad, Cys230 was found to participate in disulfide bonds with both Cys230 and its neighbor, Cys231 Notably, Ca2+ was found to protect against formation of these disulfide bonds. To investigate the role of each cysteine residue, we created alanine mutants and found that Cys230 appears to promote oxidation and inactivation of TG2 facilitating formation of Cys370-Cys371 through formation the Cys230-Cys370 disulfide bond. Although vicinal disulfide pairs are found in several transglutaminase isoforms, Cys230 is unique for TG2, suggesting that this residue acts as an isoform specific redox sensor. Our findings suggest that oxidation is likely to influence the amount of active TG2 present in the extra cellular environment. [ABSTRACT FROM AUTHOR]

γ-Glutamylamine cyclotransferase (GGACT) is an enzyme that converts γ-glutamylamines to free amines and 5-oxoproline. GGACT shows high activity toward γ-glutamyl-&3x20AC;-lysine, derived from the breakdown of fibrin and other proteins crosslinked by transglutaminases. The enzyme adopts the newly identified cyclotransferase fold, observed in γ-glutamylcyclotransferase (GGCT), an enzyme with activity toward γ-glutamyl-a-amino acids (Oakley, A. J., Yamada, T., Liu, D., Coggan, M., Clark, A. G., and Board, P. G. (2008) J. Biol. Chem. 283, 22031-22042). Despite the absence of significant sequence identity, several residues are conserved in the active sites of GGCT and GGACT, including a putative catalytic acid/base residue (GGACT Glu82). The structure of GGACT in complex with the reaction product 5-oxoproline provides evidence for a common catalytic mechanism in both enzymes. The proposed mechanism, combined with the three-dimensional structures, also explains the different substrate specificities of these enzymes. Despite significant sequence divergence, there are at least three subfamilies in prokaryotes and eukaryotes that have conserved the GGCT fold and GGCT enzymatic activity. [ABSTRACT FROM AUTHOR]

We have demonstrated previously that the Myc oncoprotein blocks cancer cell differentiation by forming a novel transcriptional repressor complex with histone deacetylase and inhibiting gene transcription of tissue transglutaminase (TG2). Moreover, induction of TG2 gene transcription and transamidase activity is essential for the differentiating effects of retinoids in cancer cells. Here, we show that two structurally distinct TG2 protein isoforms, the full-length (TG2-L) and the short form (TG2-S), exert opposing effects on cell differentiation. Repres- sion of TG2-L with small interfering RNA, which did not affect TG2-S expression, induced dramatic neuritic differentiation in neurobtastoma cells. In contrast, overexpression of TG2-S or a GTP-binding-deficient mutant of TG2-L (RS80A), both of which lack the GTP-binding Arg-580 residue, induced neuroblastoma cell differentiation, which was blocked by an inhibitor of transamidase activity. Whereas N-Myc repressed and retinoid activated both TG2 isoforms, repression of TG2-L, but not simultaneous repression of TG2-L and TG2-S, enhanced neuroblastoma cell differentiation due to N-Myc small interfering RNA or retinoid. Moreover, suppression of vasoactive intestinal peptide (VIP) expression alone induced neuroblastoma cell differentiation, and VIP was up-regulated by TG2-L, but not TG2-S. Taken together, our data indicate that TG2-L and TG2-S exert opposite effects on cell differentiation due to differences in GTP binding and modulation of VIP gene transcription. Our findings highlight the potential importance of repressing the GTP binding activity of TG2-L or activating the transamidase activity of TG2-L or TG2-S for the treatment of neuroblastoma, and possibly also other Myc-induced malignancies, and for enhancing retinoid anticancer effects. [ABSTRACT FROM AUTHOR]

Transglutaminase 2 (TG2) is a multifunctional protein that has been implicated in numerous pathologies including that of neurodegeneration and celiac disease, but the molecular interactions that mediate its diverse activities are largely unknown. Bcr and the closely related Abr negatively regulate the small G-protein Rac: loss of their combined function in vivo results in increased reactivity of innate immune cells. Bcr and Abr are GTPase-activating proteins that catalyze the hydrolysis of the GTP bound to Rac. However, how the Bcr and Abr GTPaseactivating activity is regulated is not precisely understood. We here report a novel mechanism of regulation through direct protein-protein interaction with TG2. TG2 bound to the Rac-binding pocket in the GTPase-activating domains of Bcr and Abr, blocked Bcr activity and, through this mechanism, increased levels of active GTP-bound Rac and EGF-stimulated membrane ruffling. TG2 exists in at least two different conformations. Interestingly, experiments using TG2 mutants showed that Bcr exhibits preferential binding to the non-compacted conformation of TG2, in which its catalytic domain is exposed, but transamidation is not needed for the interaction. Thus, TG2 regulates levels of cellular GTP-bound Rac and actin cytoskeletal reorganization through a new mechanism involving direct inhibition of Bcr GTPase-activating activity. [ABSTRACT FROM AUTHOR]

Sonic hedgehog (Shh) signaling plays major roles in embryonic development and has also been associated with the progression of certain cancers. Here, Shh family members act directly as long range morphogens, and their ability to do so has been linked to the formation of freely diffusible multimers from the lipidated, cell-tethered monomer (ShhNp). In this work we demonstrate that the multimeric morphogen secreted from endogenous sources, such as mouse embryos and primary chick chondrocytes, consists of oligomeric substructures that are "undisruptable" by boiling, denaturants, and reducing agents. Undisruptable (UD) morphogen oligomers vary in molecular weight and possess elevated biological activity if compared with recombinant Sonic hedgehog (ShhN). However, ShhN can also undergo UD oligomerization via a heparan sulfate (HS)-dependent mechanism in vitro, and HS isolated from different sources differs in its ability to mediate UD oligomer formation. Moreover, site-directed mutagenesis of conserved ShhN glutamine residues abolishes UD oligomerization, and inhibitors directed against transglutaminase (TG) activity strongly decrease the amount of chondrocyte-secreted UD oligomers. These findings reveal an unsuspected ability of the N-terminal hedgehog (Hh) signaling domain to form biologically active, covalently crosslinked oligomers and a novel HS function in this TG-catalyzed process. We suggest that in hypertrophic chondrocytes, HS-assisted, TG-mediated Hh oligomerization modulates signaling via enhanced protein signaling activity. [ABSTRACT FROM AUTHOR]

In fibrotic conditions increases in TG2 activity has been linked to an increase in the deposition of extracellular matrix proteins. Using TG2 transfected Swiss 313 fibroblasts expressing TG2 under the control of the tetracycline-regulated inducible promoter, we demonstrate that induction of TG2 not only stimulates an increase in collagen and fibronectin deposition but also an increase in the expression of these proteins. Increased TG2 expression in these fibroblasts led to NF-κB activation, resulting in the increased expression of transforming growth factor (TGF) β1. In addition, cells overexpressing TG2 demonstrated an increase in biologically active TGFβ1 in the extracellular environment. A specific site-directed inhibitor of TG abolished the NF-κB and TGFβ1 activation and the subsequent elevation in the synthesis and deposition of extracellular matrix proteins, confirming that this process depends on the induction of transglutaminase activity. Treatment of TG2-induced fibroblasts with nontoxic doses of nitric oxide donor S-nitroso-N-acetylpenicillamine resulted in decreased TG2 activity and apprehension of the inactive enzyme on the cell surface. This was paralleled by a reduction in activation of NE-KB and TGFβ1 production with a subsequent decrease in collagen expression and deposition. These findings support a role for NO in the regulation of TG2 function in the extracellular environment. [ABSTRACT FROM AUTHOR]

The α-synuclein immunopositive and chaotrope-insoluble material from human brains with Lewy body pathology was analyzed by mass spectrometry. From the proteinase K-cleavable peripheral fraction of Lewy bodies, which was densely cross-linked by γ-glutamyl-ϵ-lysine bonds between HspB1 and ubiquitin in a pattern similar to neurofibrillary tangles (Nemes, Z., Devreese, B., Steinert, P. M., Van Beeumen, J., and Fésüs, L. (2004) FASEB J. 18, 1135-1137), 53 proteins were identified. In the core of Lewy bodies only α-synuclein was found, and it contained a low amount of intramolecular cross-links between Gln-99 and Lys-58. In vitro cross-linking of α-synuclein by transglutaminases 1-3 and 5 produced a heterogeneous population of variably cross-linked α-synucleins in solution, which inhibited the aggregation of the protein into amyloid. However, in the presence of phosphatidylserine-rich membranes and micromolar calcium concentrations, the cross-linking by transglutaminases 1, 2, and 5 showed specificity toward the utilization of Gln-99 and Lys-58. As shown by thioflavin T fluorescence monitoring, the formation of this cross-link accelerated the aggregation of native α-synuclein. Chemical cross-linking of residues 58-99 triggered amyloid formation, whereas such bonding of residues 99 to 10 was inhibitory. Our findings reveal the pivotal role of membrane attachment and transglutaminase-mediated intermolecular cross-linking for the propagative misfolding and aggregation of α-synuclein. [ABSTRACT FROM AUTHOR]