Structural basis for the coordinated regulation of transglutaminase 3 by guanine nucleotides and calcium/magnesium

Transglutaminase 3 (TGase 3) is a member of a family of Ca2+-dependent enzymes that catalyze covalent cross-linking reactions between proteins or peptides. TGase 3 isoform is widely expressed and is important for effective epithelial barrier formation in the assembly of the cell envelope. Among the nine TGase enzyme isoforms known in the human genome, only TGase 2 is known to bind and hydrolyze GTP to GDP; binding GTP inhibits its transamidation activity but allows it to function in signal transduction. Here we present biochemical and crystallographic evidence for the direct binding of GTP/GDP to the active TGase 3 enzyme, and we show that the TGase 3 enzyme undergoes a GTPase cycle. The crystal structures of active TGase 3 with guanosine 5'-O-(thiotriphosphate) (GTPgammaS) and GDP were determined to 2.1 and 1.9 A resolution, respectively. These studies reveal for the first time the reciprocal actions of Ca2+ and GTP with respect to TGase 3 activity. GTPgammaS binding is coordinated with the replacement of a bound Ca2+ with Mg2+ and conformational rearrangements that together close a central channel to the active site. Hydrolysis of GTP to GDP results in two stable conformations, resembling both the GTP state and the non-nucleotide bound state, the latter of which allows substrate access to the active site.     

Kinetic analysis of the interaction of tissue transglutaminase with a nonpeptidic slow-binding inhibitor

Tissue transglutaminase (TGase) is a Ca2+-dependent enzyme that catalyzes cross-linking of intracellular proteins through a mechanism that involves isopeptide bond formation between Gln and Lys residues and is allosterically regulated by GTP. TGase is thought to play a pathogenic role in neurodegenerative diseases by promoting aggregation of disease-specific proteins that accumulate as part of these disorders. Given the role that TGase plays in neurodegenerative disorders, we initiated a research program to discover inhibitors of this enzyme that might ultimately be developed into therapeutic agents. To identify such inhibitors, we screened 110,000 druglike compounds for their ability to inhibit TGase [Case, A., et al. (2005) Anal. Biochem. 338, 237-244]. In this paper, we report the kinetics of interaction of human TGase with one of the inhibitors that we identified, LDN-27219. We found that this compound is a reversible, slow-binding inhibitor that appears not to bind at the enzyme's active site but rather at the enzyme's GTP site, or a site that regulates binding of GTP. Interestingly, the potency and kinetics of inhibition are dependent on substrate structure and suggest a novel mechanism of inhibition that involves differential binding of LDN-27219 to multiple conformational states of this enzyme.     

Crystal structure of transglutaminase 3 in complex with GMP: structural basis for nucleotide specificity

Epidermal-type Transglutaminase 3 (TGase 3) is a Ca(2+)-dependent enzyme involved in the cross-linking of structural proteins required in the assembly of the cell envelope. We have recently shown that calcium-activated TGase 3, like TGase 2, can bind, hydrolyze, and is inhibited by GTP despite lacking structural homology with other GTP-binding proteins. Here we report the crystal structure determined at 2.0 A resolution of TGase 3 in complex with GMP to elucidate the structural features required for nucleotide recognition. Binding affinities for various nucleotides were found by fluorescence displacement to be as follows: guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) (0.4 microm), GTP (0.6 microm), GDP (1.0 microm), GMP (0.4 microm), and ATP (28.0 microm). Furthermore, we found that GMP binds as a reversible, noncompetitive inhibitor of TGase 3 transamidation activity, similar to GTPgammaS and GDP. A genetic algorithm similarity program (GASP) approach (virtual ligand screening) identified three compounds from the Lead Quest trade mark data base (Tripos Inc.) based on superimposition of GTPgammaS, GDP, and GMP guanine nucleotides from our crystal structures to generate the minimum align flexible fragment. These three were nucleotide analogs without a phosphate group containing the minimal binding motif for TGase 3 that includes a nucleoside recognition groove. Binding affinities were measured as follows: TP349915 (K(d) = 4.1 microm), TP395289 (K(d) = 38.5 microm), TP394305 (K(d) = 1.0 mm). Remarkably, these compounds do not inhibit but instead activate TGase 3 transamidation by about 10-fold. These results suggest that the nucleotide binding pocket in TGase 3 may be exploited to either enhance or inhibit the enzymatic activity as required for different therapeutic approaches.     

Ca2+: a stabilizing component of the transglutaminase activity of Galphah (transglutaminase II)

Galphah (transglutaminase type II; tissue transglutaminase) is a bifunctional enzyme with transglutaminase (TGase) and guanosine triphosphatase (GTPase) activities. The GTPase function of Galphah is involved in hormonal signaling and cell growth while the TGase function plays an important role in apoptosis and in cross-linking extracellular and intracellular proteins. To analyze the regulation of these dual enzymatic activities we examined their calcium-dependence and thermal stability in enzymes from several cardiac sources (mouse heart, and normal, ischemic and dilated cardiomyopathic human hearts). The GTP binding activity of Galphah was markedly inhibited by Ca2+ whereas the TGase activity was strongly stimulated, suggesting that Ca2+ acts as a regulator, switching Galphah from a GTPase to a TGase. The TGase function of Galphah of both mouse and human hearts was more thermostable in the presence of Ca2+.     

GTP binding and signaling by Gh/transglutaminase II involves distinct residues in a unique GTP-binding pocket

G(h) is a dual function protein. It has receptor signaling activity that requires GTP binding and Ca(2+)-activated transglutaminase (TGase) activity that is inhibited by GTP binding. G(h) shows no homology with other GTP-binding proteins, and its GTP-binding site has not been defined. Based on sequence analysis of [alpha-(32)P]GTP-photolabeled and proteolytically released internal peptide fragments, we report localization of GTP binding to a 15-residue segment ((159)YVLTQQGFIYQGSVK(173)) of the G(h) core domain. This was confirmed by site-directed mutagenesis; a G(h)/fXIIIA chimera (in which residues 162-179 of G(h) were substituted with the equivalent but nonhomologous region of the non-GTP-binding TGase factor XIIIA) and a G(h) point mutant, S171E, retained TGase activity but failed to bind and hydrolyze GTP and did not support alpha(1B)-adrenergic receptor signaling. Slight impairment of GTP binding (1.5-fold) and hydrolysis (10-fold) in the absence of altered TGase activity did not affect signaling by the mutant K173N. However, greater impairment of GTP binding (6-fold) and hydrolysis (50-fold) abolished signaling by the mutant K173L. Mutant S171C exhibited enhanced GTP binding and signaling. Thus, residues Ser(171) and Lys(173) are critical for both GTP binding and signaling but not TGase activity. Mutagenesis of residues N-terminal to Gly(170) impaired both GTP binding and TGase activity. From computer modeling of G(h), it is evident that the GTP-binding region identified here is distinct from, but interacts with, the TGase active site. Together with structural considerations of G(h) versus other GTP-binding proteins, these findings indicate that G(h) has a unique GTP-binding pocket and provide for the first time a mechanism for GTP-mediated regulation of the TGase activity of G(h).     

Localization of cellular transglutaminase on the extracellular matrix after wounding: characteristics of the matrix bound enzyme.

Extending our previous observation that tissue transglutaminase (TGase) binds to extracellular matrix (ECM) fibronectin, we report here that endogenous tissue TGase is localized on the adjacent ECM after puncture wounding embryonic human lung fibroblasts (WI-38). The bound TGase persisted at the wound site for many hours, demonstrated by immunofluorescence and by catalytic activity using an overlay assay. The binding characteristics of TGase with ECM were studied further by the addition of exogenous TGase to cell monolayers and monitoring by immunofluorescence or overlay catalytic activity assays. Binding occurred equally well at 4 degrees C or 37 degrees C. Prior incubation of exogenous TGase with guanosine 5'-triphosphate (GTP), guanosine 5'-diphosphate (GDP), or adenosine triphosphate (ATP) had little effect on the amount bound to matrix, but prior treatment with calcium, magnesium, strontium, or manganese ions enhanced binding 2- to 3-fold. The Ca(++)-dependent change was a concentration-dependent effect on soluble exogenous TGase, rather than an effect on ECM. Immunofluorescent techniques showed that binding of exogenous TGase to ECM was prevented by prior mixing with fibronectin or collagen, but not with several other ECM components, including laminin, elastin, chondroitin sulfate, heparan sulfate, and hyaluronic acid. ECM-bound TGase was released by 2 M potassium thiocyanate (KSCN) treatment but was not released by treatment with a variety of amino acids, salts, reducing agents, glycerol, or other chaotropic agents.     

Osmotic resistance of high-density erythrocytes in transglutaminase 2-deficient mice

Transglutaminase 2 (TGase 2) is a Ca(2+)-dependent enzyme responsible for the posttransttranslational modification of proteins by transamidation of specific polypeptide-bound glutamine residues. Elevating the intracellular concentration of Ca(2+)-ions in human erythrocytes leads to the formation of cytoskeletal and cytoplasmic protein polymers. The Ca(2+)-dependent TGase 2-dependent cross-linking activity has been proposed for its involvement in erythrocyte aging, by inducing irreversible modification of their cell shape and deformability. Accordingly, we found that high-density ("old") TGase 2(minus sign/minus sign) red blood cells (RBCs) were more resistant to osmotic stress-induced hemolysis than those from wild type mice. In addition, elevating the intracellular concentration of Ca(2+) by treatment of total RBCs with ionophore A23187 resulted in enhanced resistance of TGase 2-deficient erythrocytes compared to their normal counterpart. These findings indicate that TGase 2 may have a role in regulating structural flexibility of RBCs, possibly affecting their life span in physiopathological conditions, such as erythrocyte senescence, which are accompanied by increases in intracellular Ca(2+) concentration.     

Three-dimensional structure of the human transglutaminase 3 enzyme: binding of calcium ions changes structure for activation

Transglutaminase (TGase) enzymes catalyze the formation of covalent cross-links between protein-bound glutamines and lysines in a calcium-dependent manner, but the role of Ca(2+) ions remains unclear. The TGase 3 isoform is widely expressed and is important for epithelial barrier formation. It is a zymogen, requiring proteolysis for activity. We have solved the three-dimensional structures of the zymogen and the activated forms at 2.2 and 2.1 A resolution, respectively, and examined the role of Ca(2+) ions. The zymogen binds one ion tightly that cannot be exchanged. Upon proteolysis, the enzyme exothermally acquires two more Ca(2+) ions that activate the enzyme, are exchangeable and are functionally replaceable by other lanthanide trivalent cations. Binding of a Ca(2+) ion at one of these sites opens a channel which exposes the key Trp236 and Trp327 residues that control substrate access to the active site. Together, these biochemical and structural data reveal for the first time in a TGase enzyme that Ca(2+) ions induce structural changes which at least in part dictate activity and, moreover, may confer substrate specificity.     

Ca2+ oscillations mediated by exogenous GTP in Paramecium cells: assessment of possible Ca2+ sources

We applied exogenous guanosine trisphosphate, GTP, to Paramecium tetraurelia cells injected with Fura Red for analysing changes of free intracellular Ca(2+) concentrations, [Ca(2+)](i), during periodic back-/forward swimming thus induced. Strain ginA (non-responsive to GTP) shows no Ca(2+) signal upon GTP application. In strain nd6 (normal Ca(2+) signalling) an oscillating [Ca(2+)](i) response with a prominent first peak occurs upon GTP stimulation, but none after mock-stimulation or after 15 min adaptation to GTP. While this is in agreement with previous electrophysiological analyses, we now try to identify more clearly the source(s) of Ca(2+). Stimulation of nd6 cells, after depletion of Ca(2+) from their cortical stores (alveolar sacs), shows the same Ca(2+) oscillation pattern but with reduced amplitudes, and a normal behavioural response is observed. Stimulation with GTP, supplemented with the Ca(2+) chelator BAPTA, results in loss of the first prominent Ca(2+) peak, in reduction of the following Ca(2+) amplitudes, and in the absence of any behavioural response. Both these observations strongly suggest that for the initiation of GTP-mediated back-/forward swimming Ca(2+) from the extracellular medium is needed. For the maintenance of the Ca(2+) oscillations a considerable fraction must come from internal stores, probably other than alveolar sacs, rather likely from the endoplasmic reticulum.     

The emerging structural understanding of transglutaminase 3

Transglutaminases (TGase; protein-glutamine: amine gamma-glutamyl-transferase) are a family of calcium-dependent acyl-transfer enzymes ubiquitously expressed in mammalian cells and responsible for catalyzing covalent cross-links between proteins or peptides. A series of recent crystal structures have revealed the overall architecture of TGase enzymes, and provided a deep look at their active site, calcium and magnesium ions, and the manner by which guanine nucleotides interact with this enzyme. These structures, backed with extensive biochemical studies, are providing new insights as to how access to the enzyme's active site may be gated through the coordinated changes in cellular calcium and magnesium concentrations and GTP/GDP. Calcium-activated TGase 3 can bind, hydrolyze, and is inhibited by GTP, despite lacking structural homology with other GTP binding proteins. A structure based sequence homology among the TGase enzyme family shows that these essential structural features are shared among other members of the TGase family.     

Leishmania species: evidence for transglutaminase activity and its role in parasite proliferation

Albeit transglutaminase (TGase) activity has been reported to play crucial physiological roles in several organisms including parasites; however, there was no previous report(s) whether Leishmania parasites exhibit this activity. We demonstrate herein that TGase is functionally active in Leishmania parasites by using labeled polyamine that becomes conjugated into protein substrates. The parasite enzyme was about 2- to 4-fold more abundant in Old World species than in New World ones. In L. amazonensis, comparable TGase activity was found in both promastigotes and amastigotes. TGase activity in either parasite stage was optimal at the basic pH, but the enzyme in amastigote lysates was more stable at higher temperatures (37-55 degrees C) than that in promastigote lysates. Leishmania TGase differs from mouse macrophage (M Phi) TGase in two ways: (1) the parasite enzyme is Ca(2+)-independent, whereas the mammalian TGase depends on the cation for activity, and (2) major protein substrates for L. amazonensis TGase were found within the 50-75 kDa region, while those for the M Phi TGase were located within 37-50 kDa. The potential contribution of TGase-catalyzed reactions in promastigote proliferation was supported by findings that standard inhibitors of TGase [e.g., monodansylcadaverine (MDC), cystamine (CS), and iodoacetamide (IodoA)], but not didansylcadaverine (DDC), a close analogue of MDC, had a profound dose-dependent inhibition on parasite growth. Myo-inositol-1-phosphate synthase and leishmanolysin (gp63) were identified as possible endogenous substrates for L. amazonensis TGase, implying a role for TGase in parasite growth, development, and survival.     

GTP, an inhibitor of transglutaminases, is hydrolyzed by tissue-type transglutaminase (TGase 2) but not by epidermal-type transglutaminase (TGase 3)

Epidermal-type transglutaminase (TGase 3) is devoid of GTPase activity, but its TGase activity is inhibited by GTP as in the case of tissue-type TGase (TGase 2). In addition, the inhibition was not affected by the presence of higher concentrations of Ca ion. These results indicate that GTP interacts with TGase 3 in a manner different from its action on TGase 2.     

GTP is required to stabilize and display transamidation activity of transglutaminase 2

Transglutaminase 2 (TGase 2) is a bifunctional enzyme that catalyzes calcium-dependent transamidation and GTP binding/hydrolysis. The transamidation activity is proposed to be associated with several neurodegenerative disorders such as Alzheimer's and Hungtinton's disease. However, the regulation mechanism by which TGase 2 causes neurodegeneration is unknown. In this study, we show that two activities of TGase 2 have a differential stability; transamidation activity is less stable than GTP hydrolytic activity, and that GTP was required to stabilize and to display transamidation activity. Moreover, GTP binding-defective mutant of TGase 2 did not show any transamidation activity in transfection experiments. These results indicate that GTP binding is crucial for transamidation activity of TGase 2, suggesting that protein cross-linking by TGase 2 might be associated with G-protein coupled receptor signaling system. Thus, our data could contribute to understand the regulation of TGase 2 activity and TGase 2-associated pathogenesis.     

Regulatory properties of magnesium-dependent guanylate cyclase in Dictyostelium discoideum membranes

We have characterized a magnesium-dependent guanylate cyclase in homogenates of Dictyostelium discoideum cells. 1) The enzyme shows an up to 4-fold higher cGMP synthesis in the presence of GTP analogues with half-maximal activation at about 1 microM guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) or 100 microM guanosine 5'-(beta, gamma-imido)triphosphate; little or no stimulation was observed with GTP, guanosine mono- and diphosphates or with adenine nucleotides, with the exception of the ATP analogue adenosine 5'-(beta, gamma-imido)triphosphate. 2) Both basal and GTP gamma S-stimulated guanylate cyclase activity were rapidly lost from homogenates as was the ability of GTP gamma S to stimulate the enzyme after cell lysis. 3) Inclusion of 25 microM GTP gamma S during cell lysis reduced the KM for GTP from 340 to 85 microM and increased the Vmax from 120 to 255 pmol/min.mg protein, as assayed in homogenates 90 s after cell lysis. 4) Besides acting as an activator, GTP gamma S was also a substrate for the enzyme with a KM = 120 microM and a Vmax = 115 pmol/min.mg protein. 5) GTP gamma S-stimulated, Mg2+-dependent guanylate cyclase was inhibited by submicromolar concentrations of Ca2+ ions, and by inositol 1,4,5-trisphosphate in the absence of Ca2+ chelators. 6) Guanylate cyclase activity was detected in both supernatant and pellet fractions after 1 min centrifugation at 10,000 x g; however, only sedimentable enzyme was stimulated by GTP gamma S. We suggest that the Mg2+-dependent guanylate cyclase identified represents the enzyme that in intact cells is regulated via cell surface receptors, and we propose that guanine nucleotides are allosteric activators of this enzyme and that Ca2+ ions play a role in the maintenance of the enzyme in its basal state.     

Phosphoinositide 3-kinase activity is required for retinoic acid-induced expression and activation of the tissue transglutaminase

Tissue transglutaminase (TGase) is a dual function enzyme that couples an ability to bind GTP with transamidation activity. Retinoic acid (RA) consistently induces TGase expression and activation, and it was recently shown that increased TGase expression protected cells from apoptosis. To better understand how RA regulates TGase, we considered whether RA employed pro-survival signaling pathways to mediate TGase expression and activation. It was found that RA stimulation of NIH3T3 cells activated ERK and phosphoinositide 3-kinase (PI3K); however, only PI3K activation was necessary for RA-induced TGase expression. The overexpression of a constitutively active form of PI3K did not induce TGase expression, indicating that PI3K signaling was necessary but not sufficient for TGase expression. The exposure of cells expressing exogenous TGase to the PI3K inhibitor, LY294002, reduced the ability of TGase to be photoaffinity-labeled with [alpha-(32)P]GTP, providing evidence that PI3K regulates the GTP binding activity of TGase as well as its expression. Moreover, cell viability assays showed that incubation of RA-treated cells with LY294002 together with the TGase inhibitor, monodansylcadaverine (MDC), converted RA from a differentiation factor to an apoptotic stimulus. These findings demonstrate that PI3K activity is required for the RA-stimulated expression and GTP binding activity of TGase, thereby linking the up-regulation of TGase with a well established cell survival factor.     

Protein transamidation by transglutaminase 2 in cells: a disputed Ca2+-dependent action of a multifunctional protein

Transglutaminase 2 (TG2) is the first described cellular member of an enzyme family catalyzing Ca(2+)-dependent transamidation of proteins. During the last two decades its additional enzymatic (GTP binding and hydrolysis, protein disulfide isomerase, protein kinase) and non-enzymatic (multiple interactions in protein scaffolds) activities, which do not require Ca(2+) , have been recognized. It became a prevailing view that TG2 is silent as a transamidase, except in extreme stress conditions, in the intracellular environment characterized by low Ca(2+) and high GTP concentrations. To counter this presumption a critical review of the experimental evidence supporting the role of this enzymatic activity in cellular processes is provided. It includes the structural basis of TG2 regulation through non-canonical Ca(2+) binding sites, mechanisms making it sensitive to low Ca(2+) concentrations, techniques developed for the detection of protein transamidation in cells and examples of basic cellular phenomena as well as pathological conditions influenced by this irreversible post-translational protein modification.     

Identification of ML204, a novel potent antagonist that selectively modulates native TRPC4/C5 ion channels

Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable nonselective cation channels implicated in diverse physiological functions, including smooth muscle contractility and synaptic transmission. However, lack of potent selective pharmacological inhibitors for TRPC channels has limited delineation of the roles of these channels in physiological systems. Here we report the identification and characterization of ML204 as a novel, potent, and selective TRPC4 channel inhibitor. A high throughput fluorescent screen of 305,000 compounds of the Molecular Libraries Small Molecule Repository was performed for inhibitors that blocked intracellular Ca(2+) rise in response to stimulation of mouse TRPC4β by μ-opioid receptors. ML204 inhibited TRPC4β-mediated intracellular Ca(2+) rise with an IC(50) value of 0.96 μm and exhibited 19-fold selectivity against muscarinic receptor-coupled TRPC6 channel activation. In whole-cell patch clamp recordings, ML204 blocked TRPC4β currents activated through either μ-opioid receptor stimulation or intracellular dialysis of guanosine 5'-3-O-(thio)triphosphate (GTPγS), suggesting a direct interaction of ML204 with TRPC4 channels rather than any interference with the signal transduction pathways. Selectivity studies showed no appreciable block by 10-20 μm ML204 of TRPV1, TRPV3, TRPA1, and TRPM8, as well as KCNQ2 and native voltage-gated sodium, potassium, and calcium channels in mouse dorsal root ganglion neurons. In isolated guinea pig ileal myocytes, ML204 blocked muscarinic cation currents activated by bath application of carbachol or intracellular infusion of GTPγS, demonstrating its effectiveness on native TRPC4 currents. Therefore, ML204 represents an excellent novel tool for investigation of TRPC4 channel function and may facilitate the development of therapeutics targeted to TRPC4.     

Involvement of pertussis toxin-sensitive and -insensitive GTP-binding proteins in luteinizing hormone exocytosis distal to second messenger generation.

Inhibition of luteinizing hormone (LH) exocytosis by guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) in permeabilized pituitary cells has indicated the involvement of one or more GTP-binding proteins in the exocytotic mechanism distal to second messenger generation. We now report that two inhibitory sites of action of GTP gamma S can be distinguished by their dependence on GTP gamma S concentration and their sensitivity to pertussis toxin. Ca(2+)-stimulated exocytosis was half-maximally inhibited by 6.8 microM GTP gamma S, a six-fold higher concentration than that required for inhibition of exocytosis stimulated by phorbol ester plus cAMP. In addition, GTP gamma S inhibition of Ca(2+)-stimulated exocytosis was insensitive to pertussis toxin, in contrast to the inhibition of exocytosis stimulated by phorbol ester plus cAMP, which was abolished by pretreatment with pertussis toxin. These results indicate that at least two stimulus-specific GTP-binding proteins are involved in regulating LH exocytosis distal to second messenger generation.