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.     

Serotonylated fibronectin is elevated in pulmonary hypertension

Serotonin (5-HT) and fibronectin (FN) have been associated with pulmonary hypertension (PH). We previously reported that FN is posttranslationally modified by tissue transglutaminase (TGase) to form serotonylated FN (s-FN) in pulmonary artery smooth muscle cells and that serotonylation stimulates their proliferation and migration, hallmarks of PH. We hypothesized that s-FN and its binding to TGase are elevated in human and experimental PH. To assess this hypothesis, FN isolation and electrophoretic, immunoblotting, and densitometric techniques were used. Mean ratio of serum s-FN to total FN level (s-FN/FN) was elevated in 19 consecutive pulmonary arterial hypertension (PAH) patients compared with 25 controls (0.3 ± 0.18 vs. 0.05 ± 0.07, P < 0.001). s-FN/FN also was increased in lungs of mice and rats with hypoxia-induced PH and in rats with monocrotaline-induced PH. In mice, the increase was detected at 1 wk of hypoxia, preceding the development of PH. Hypoxic rats had elevated serum s-FN/FN. Enhanced binding of TGase to its substrate FN occurred in serum from patients with PAH (mean 0.50 ± 0.51 vs. 0.063 ± 0.11, P = 0.002) and s-FN/FN and TGase-bound FN were highly correlated (R(2) = 0.77). TGase-bound FN also was increased in experimental PH. We conclude that increased serotonylation of FN occurs in human and experimental PH and may provide a biomarker for the disease.     

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.     

Cloning and characterization of novel tumor-targeting immunocytokines based on murine IL7

We generated and characterized novel antibody-cytokine fusion proteins (“immunocytokines”) based on murine interleukin-7 (IL7), an immunomodulatory protein which has previously shown anti-cancer activity in preclinical models and whose human counterpart is currently being investigated in clinical trials. The sequential fusion of the clinical-stage antibody fragment scFv(F8), specific to a tumor-associated splice isoform of fibronectin, yielded an immunocytokine (termed “F8-mIL7”) of insufficient pharmaceutical quality and in vivo tumor targeting performance, with a striking dose dependence on tumor targeting selectivity. By contrast, a novel immunocytokine design (termed “F8-mIL7-F8”), in which two scFv moieties were fused at the N- and C-terminus of murine IL7, yielded a protein of excellent pharmaceutical quality and with improved tumor-targeting performance [tumor: blood ratio = 16:1, 24 h after injection]. Both F8-mIL7 and F8-mIL7-F8 could induce tumor growth retardation in immunocompetent mice, but were not able to eradicate F9 tumors. The combination of F8-mIL7-F8 with paclitaxel led to improved therapeutic results, which were significantly better compared to those obtained with saline treatment. The study indicates how the engineering of novel immunocytokine formats may help generate fusion proteins of acceptable pharmaceutical quality, for those immunomodulatory proteins which do not lend themselves to a direct fusion with antibody fragments.     

Effects of Glycine on the Catecholamine Levels and Activities of Alcohol-Metabolizing Enzymes in Rats with Alcohol Intoxication and Addiction

We studied in rats the effects of peroral glycine introduction on the contents of catecholamines (CA) – noradrenaline (NA) and dopamine (DA) – in different brain structures (hypothalamus, midbrain, and neocortex), as well as the levels of adrenaline (A), NA, and DA in the blood and the activity of alcohol-metabolizing (AlM) enzymes – alcohol dehydrogenase (AlDH) and aldehyde dehydrogenase (AdhDH) – in the blood serum. The experimental group included animals with a disposition to alcohol consumption under conditions of free choice for drinking between an alcohol solution and water. The measurements were performed in animals in the state of acute alcohol intoxication (i.p. injection of 4 g/kg ethanol) or chronic alcohol addiction (formed due to a 3-month-long free access to ethanol solution). Introduction of 150 mg/kg glycine increased the NA and DA contents (the latter, to a lesser extent) in all examined brain structures; the NA level in the blood increased, while that of DA decreased. Under conditions of acute alcohol intoxication and chronic alcohol addiction, the ratio of the activities of AlM enzymes, AdhDH/AlDH, was significantly shifted toward values indicative of accumulation of acetaldehyde (AcAdh) in the tissues. This was accompanied by noticeable modifications of the CA contents in the brain structures and blood of the rats; in particular, the [DA]/[NA] ratio in the brain significantly increased. Introduction of glycine under conditions of acute alcohol intoxication provided normalization of the AdhDH/AlDH activity ratio. Obvious trends toward normalization of the CA levels in the brain structures were also observed in both acute and chronic experiments. In the latter case, the glycine treatment course resulted in a drop in the daily alcohol consumption by the animals. We conclude that glycine, which binds AcAdh and modifies the metabolism of CA transmitters, exerts a significant corrective influence on the pathogenetic mechanisms of alcohol addiction. Our experimental findings demonstrate that there are contact points between the acetaldehyde and catecholamine hypotheses of pathogenesis of alcoholism.     

Screening for the preferred substrate sequence of transglutaminase using a phage-displayed peptide library: identification of peptide substrates for TGASE 2 and Factor XIIIA

Mammalian transglutaminase (TGase) catalyzes covalent cross-linking of peptide-bound lysine residues or incorporation of primary amines to limited glutamine residues in substrate proteins. Using an unbiased M13 phage display random peptide library, we developed a screening system to elucidate primary structures surrounding reactive glutamine residue(s) that are preferred by TGase. Screening was performed by selecting phage clones expressing peptides that incorporated biotin-labeled primary amine by the catalytic reactions of TGase 2 and activated Factor XIII (Factor XIIIa). We identified several amino acid sequences that were preferred as glutamine donor substrates, most of which have a marked tendency for individual TGases: TGase 2, QxPphiD(P), QxPphi, and QxxphiDP; Factor XIIIa, QxxphixWP (where x and phi represent a non-conserved and a hydrophobic amino acid, respectively). We further confirmed that the sequences were favored for transamidation using modified glutathione S-transferase (GST) for recombinant peptide-GST fusion proteins. Most of the fusion proteins exhibited a considerable increase in incorporation of primary amines over that of modified GST alone. Furthermore, we identified the amino acid sequences that demonstrated higher specificity and inhibitory activity in the cross-linking reactions by TGase 2 and Factor XIIIa.     

Identification of mammalian-type transglutaminase in Physarum polycephalum. Evidence from the cDNA sequence and involvement of GTP in the regulation of transamidating activity.

Transglutaminase (TGase) catalyses the post-translational modification of proteins by transamidation of available glutamine residues. While several TGase genes of fish and arthropods have been cloned and appear to have similar structures to those of mammals, no homologous gene has been found in lower eukaryotes. We have cloned the acellular slime mold Physarum polycephalum TGase cDNA using RT-PCR with degenerated primers, based on the partial amino acid sequence of the purified enzyme. The cDNA contained a 2565-bp ORF encoding a 855-residue polypeptide. By Northern blotting, an mRNA of approximately 2600 bases was detected. In comparison with primary sequences of mammalian TGases, surprisingly, significant similarity was observed including catalytic triad residues (Cys, His, Asn) and a GTP-binding region. The alignment of sequences and a phylogenetic tree also demonstrated that the structure of P. polycephalum TGase is similar to that of TGases of vertebrates. Furthermore, we observed that the purified TGase had GTP-hydrolysing activity and that GTP inhibited its transamidating activity, as in the case of mammalian tissue-type TGase (TGase 2).     

Development of an isoenzyme-specific colorimetric assay for tissue transglutaminase 2 cross-linking activity

Tissue transglutaminase (TGase 2) belongs to the multigene transglutaminase family of Ca²⁺-dependent protein cross-linking enzymes. Based on the transamidation activity of TGase 2, a novel colorimetric assay has been developed using covalently coupled spermine to carboxy-substituted polystyrene plates and biotinylated pepT26, an excellent acyl-donor substrate, highly specific for TGase 2. The assay is based on the incorporation of the gamma-carboxamide of glutamine of pepT26 into the immobilized spermine. The amount of biotinylated pepT26 bound to the plate, as measured by the activity of streptavidin-peroxidase, is directly proportional to the TGase activity. The colorimetric procedure showed a good correlation (r = 0.995) with the commonly used radiometric filter paper method for TGase2, and provides linear dose-response curves over a wide range of hrTGase2 concentrations (2.5-40 μU/ml). In addition, the assay shows higher sensitivity when compared with our previous TG-colorimetric test (more than 50-fold increase) and other existing assays. PepT26 displays strong reactivity with TGase 2, and no reactivity with TGases 1, 3, and FXIII. The procedure constitutes a rapid, TG2-specific, sensitive, and nonisotopic method for the measurement of TGase 2 activity in as low as 4 ng of hrTGase 2 and purified guinea pig liver transglutaminase, and 1.25 μg of guinea pig liver extracts.     

The state transition mechanism—simply depending on light-on and -off in Spirulina platensis

The state transition in cyanobacteria is a long-discussed topic of how the photosynthetic machine regulates the excitation energy distribution in balance between the two photosystems. In the current work, whether the state transition is realized by “mobile phycobilisome (PBS)” or “energy spillover” has been clearly answered by monitoring the spectral responses of the intact cells of the cyanobacterium Spirulina platensis. Firstly, light-induced state transition depends completely on a movement of PBSs toward PSI or PSII while the redox-induced one on not only the “mobile PBS” but also an “energy spillover”. Secondly, the “energy spillover” is triggered by dissociation of PSI trimers into the monomers which specially occurs under a case from light to dark, while the PSI monomers will re-aggregate into the trimers under a case from dark to light, i.e., the PSI oligomerization is reversibly regulated by light switch on and off. Thirdly, PSI oligomerization is regulated by the local H⁺ concentration on the cytosol side of the thylakoid membranes, which in turn is regulated by light switch on and off. Fourthly, PSI oligomerization change is the only mechanism for the “energy spillover”. Thus, it can be concluded that the “mobile PBS” is a common rule for light-induced state transition while the “energy spillover” is only a special case when dark condition is involved.     

Role of transglutaminase II in retinoic acid-induced activation of RhoA-associated kinase-2

Transamidation is a post-translational modification of proteins mediated by tissue transglutaminase II (TGase), a GTP-binding protein, participating in signal transduction pathways as a non-conventional G-protein. Retinoic acid (RA), which is known to have a role in cell differentiation, is a potent activator of TGASE: The activation of TGase results in increased transamidation of RhoA, which is inhibited by monodansylcadaverine (MDC; an inhibitor of transglutaminase activity) and TGaseM (a TGase mutant lacking transglutaminase activity). Transamidated RhoA functions as a constitutively active G-protein, showing increased binding to its downstream target, RhoA-associated kinase-2 (ROCK-2). Upon binding to RhoA, ROCK-2 becomes autophosphorylated and demonstrates stimulated kinase activity. The RA-stimulated interaction between RhoA and ROCK-2 is blocked by MDC and TGaseM, indicating a role for transglutaminase activity in the interaction. Biochemical effects of TGase activation, coupled with the formation of stress fibers and focal adhesion complexes, are proposed to have a significant role in cell differentiation.     

The sulfhydryl oxidase Erv1 is a substrate of the Mia40-dependent protein translocation pathway

The thiol oxidase Erv1 and the redox-regulated receptor Mia40/Tim40 are components of a disulfide relay system which mediates import of proteins into the intermembrane space (IMS) of mitochondria. Here we report that Erv1 requires Mia40 for its import into mitochondria. After passage across the translocase of the mitochondrial outer membrane Erv1 interacts via disulfide bonds with Mia40. Erv1 does not contain twin “CX₃C” or twin “CX₉C” motifs which are crucial for import of typical substrates of this pathway and it does not need two “CX₂C” motifs for import into mitochondria. Thus, Erv1 represents an unusual type of substrate of the Mia40-dependent import pathway.     

Thiol reactivity as a sensor of rotation of the converter in myosin

Smooth muscle myosin has two reactive thiols located near the C-terminal region of its motor domain, the “converter”, which rotates by ∼70° upon the transition from the “nucleotide-free” state to the “pre-power stroke” state. The incorporation rates of a thiol reagent, 5-(((2-iodoacetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid (IAEDANS), into these thiols were greatly altered by adding ATP or changing the myosin conformation. Comparisons of the myosin structures in the pre-power stroke state and the nucleotide-free state explained why the reactivity of both thiols is especially sensitive to a conformational change around the converter, and thus can be used as a sensor of the rotation of the converter. Modeling of the myosin structure in the pre-power stroke state, in which the most reactive thiol, “SH1”, was selectively modified with IAEDANS, revealed that this label becomes an obstacle when the converter completely rotates toward its position in the pre-power stroke state, thus resulting in incomplete rotation of the converter. Therefore, we suggest that the limitation of the converter rotation by modification causes the as-yet unexplained phenomena of SH1-modified myosin, including the inhibition of 10S myosin formation and the losses in phosphorylation-dependent regulation of the basic and actin-activated Mg-ATPase activities of myosin.     

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.     

Cystamine prevents ischemia-reperfusion injury by inhibiting polyamination of RhoA

Transglutaminase2 (TGase2) activates Rho-associated kinase (ROCK), an important mediator of ischemia-reperfusion (IR) injury, through polyamination of RhoA. Cystamine, an oxidized dimer of cysteamine inhibits the transamidation activity of TGase2. We examined whether addition of cystamine to an organ preservation solution protects rat cardiomyocyte cells (H9C2) from cell death in IR injury. H9C2 cells were stored under hypoxic conditions at 4 °C in laboratory-made preservation solution (SNU) or SNU solution supplemented with cystamine (SNU-C1), and cell preservation in the two solutions was compared by measuring the release of lactate dehydrogenase. The cells were preserved more effectively in SNU-C1 than in SNU solution. Cystamine inhibited the intracellular activity of TGase2 which increased during cold storage or reoxygenation. The inhibition of TGase2 by cystamine reduced the polyamination of RhoA, the interaction between RhoA and ROCK2, and F-actin formation. Cystamine also prevented the activation of caspases during cold storage. These results suggest that addition of cystamine to the organ preservation solution significantly enhances cardiomyocytes preservation apparently by inhibiting TGase2-mediated RhoA-ROCK pathway and that TGase2 may play an important role in IR injury by regulating ROCK.     

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.     

The Capsid Proteins of a Large, Icosahedral dsDNA Virus

Chilo iridescent virus (CIV) is a large (∼ 1850 Å diameter) insect virus with an icosahedral, T = 147 capsid, a double-stranded DNA (dsDNA) genome, and an internal lipid membrane. The structure of CIV was determined to 13 Å resolution by means of cryoelectron microscopy (cryoEM) and three-dimensional image reconstruction. A homology model of P50, the CIV major capsid protein (MCP), was built based on its amino acid sequence and the structure of the homologous Paramecium bursaria chlorella virus 1 Vp54 MCP. This model was fitted into the cryoEM density for each of the 25 trimeric CIV capsomers per icosahedral asymmetric unit. A difference map, in which the fitted CIV MCP capsomers were subtracted from the CIV cryoEM reconstruction, showed that there are at least three different types of minor capsid proteins associated with the capsomers outside the lipid membrane. “Finger” proteins are situated at many, but not all, of the spaces between three adjacent capsomers within each trisymmetron, and “zip” proteins are situated between sets of three adjacent capsomers at the boundary between neighboring trisymmetrons and pentasymmetrons. Based on the results of segmentation and density correlations, there are at least eight finger proteins and three dimeric and two monomeric zip proteins in one asymmetric unit of the CIV capsid. These minor proteins appear to stabilize the virus by acting as intercapsomer cross-links. One transmembrane “anchor” protein per icosahedral asymmetric unit, which extends from beneath one of the capsomers in the pentasymmetron to the internal leaflet of the lipid membrane, may provide additional stabilization for the capsid. These results are consistent with the observations for other large, icosahedral dsDNA viruses that also utilize minor capsid proteins for stabilization and for determining their assembly.     

Characterization and polymorphism of Keratin Associated Protein 1.4 gene in goats

Keratin-associated proteins (KAPs) are among the main structural components of the animal fibers and form semi-rigid matrix wherein the keratin intermediate filaments (KIFs) are embedded. Variation in the KAP genes has been reported to affect the structure of KAPs and hence fiber characteristics. As no information is available on this gene in Capra hircus therefore, present work was undertaken to characterize and explore the different polymorphic variants of KAP1.4 gene at DNA level in different breeds/genetic groups of goats of Kashmir. Cashmere (Changthangi, 30 animals) and non-Cashmere (Bakerwal and Kargil goats, 20 animals each) goats formed the experimental animals for the study. Single strand conformation polymorphism technique was employed for exploring variability at gene level. On exploring the size variability in KAP1.4 gene between Ovine and Caprine, it was concluded that sheep KAP1.4 gene has a deletion of 30 nucleotides. In comparison to published nucleotide sequences of sheep, goat sequences explored are differing at positions 174, 462 and 568 and at these positions “G”, “T” and “T” nucleotides are present in sheep, but are replaced by “A”, “C” and “C” respectively, in goats. By SSC studies, two genotypes were observed in each genetic group and in Bakerwal goats the genotypes were designated as A1A1 (0.40) and A1A2 (0.60) and were formed by two alleles A1 (0.70) andA2 (0.30). The different SSC patterns observed in Kargil goats were designated as B1B1 (0.35) and B1B2 (0.65) genotypes with frequencies of B1 and B2 alleles as 0.675 and 0.325, respectively. Similarly, two genotypes C1C1 (0.60) and C1C2 (0.40) were observed in Changthangi goats and the frequencies of C1 and C2 alleles were 0.80 and 0.20, respectively. These alleles were later confirmed by sequencing. The sequences of these alleles are available in NCBI under Acc. No's. JN012101.1, JN012102.1, JN000317.1, JN000318.1, JQ436929 and JQ627657. It was concluded that all the alleles observed in a breed were unique to the breed. The designated A1 and A2 alleles of Bakerwal goats differ from each other at positions 245 and the nucleotides observed were “C” or “A” and at position 605 of the nucleotide sequence “T” or “C”, were observed. The designated B1 and B2 alleles of Kargil goats differed from each other at positions 224, 374, 375 and 521. The nucleotides observed in two SSC pattern were C→G, A→G, G→A and T→C, respectively. The designated C1 and C2 alleles of Changthangi goats differed from each other at one position 440 with the change of “A”→“C”.     

Development of a mechanism-based assay for tissue transglutaminase--results of a high-throughput screen and discovery of inhibitors

Tissue transglutaminase (TGase) is a Ca(2+)-dependent enzyme that catalyzes cross-linking of intracellular proteins through a mechanism that involves isopeptide bond formation between Gln and Lys residues. In addition to its transamidation activity, TGase can bind guanosine 5'-triphosphate (GTP) and does so in a manner that is antagonized by calcium. Once bound, GTP undergoes hydrolysis to form guanosine 5'-diphosphate and inorganic phosphate. TGase is thought to play a pathogenic role in neurodegenerative diseases by promoting aggregation of disease-specific proteins that accumulate in these disorders. Thus, this enzyme represents a viable target for drug discovery. We now report the development of a mechanism-based assay for TGase and the results of a screen using this assay in which we tested 56,500 drug-like molecules for their ability to inhibit TGase. In this assay, the Gln- and Lys-donating substrates are N,N-dimethylated casein (NMC) and N-Boc-Lys-NH-CH(2)-CH(2)-NH-dansyl (KXD), respectively. Through a combination of steady state kinetic experiments and reaction progress curve simulations, we were able to calculate values for the initial concentrations of NMC, KXD, and Ca(2+) that would produce a steady state situation in which all thermodynamically significant forms of substrate-bound TGase exist in equal concentration. Under these conditions, the assay is sensitive to both competitive and mixed active-site inhibitors and to inhibitors that bind to the GTP site. The assay was optimized for automated screening in 384-well format and was then used to test our compound library. From among these compounds, 104 authentic hits that represent several mechanistic classes were identified.     

Tissue transglutaminase-mediated glutamine deamidation of beta-amyloid peptide increases peptide solubility, whereas enzymatic cross-linking and peptide fragmentation may serve as molecular triggers for rapid peptide aggregation

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 (Aβ), resulting in structural changes of monomeric Aβ. Using high resolution mass spectrometry (MS) of cross-linked Aβ peptides, 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.