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PubMed1998

Identification of transglutaminase-reactive residues in S100A11.

The recent finding that S100A11 is a component of the keratinocyte cornified envelope (CE) (Robinson, N. A., Lapic, S., Welter, J. F., and Eckert, R. L. (1997) J. Biol. Chem. 272, 12035-12046) suggests that S100A11 is a transglutaminase (TG) substrate. In the present study we show that S100A11 forms multimers when cultured keratinocytes are challenged by increased levels of intracellular calcium and that multimer formation is inhibited by the TG inhibitor, cystamine. These S100A11 multimers appear to be incorporated into the CE, as immunoreactive S100A11 is detected in purified envelopes prepared from cultured cells and from foreskin epidermis. To study S100A11 as a transglutaminase substrate, recombinant human S100A11 (rhS100A11) was used in a cell-free cross-linking system. [14C]Putrescine, a primary amine, labels rhS100A11 in a TG-dependent manner. Trypsin digestion of [14C]putrescine-labeled rhS100A11 releases one radiolabeled peptide, Ala98-Lys103. The glutamine residue in this segment, Gln102, is the site of radiolabel incorporation indicating that Gln102 functions as an amine acceptor. The ability of S100A11 to form multimers indicates that it also has a reactive lysine residue that functions as an amine donor. To identify the reactive residue, we compared the high pressure liquid chromatography profile of trypsin-digested rhS100A11 monomer to that of cross-linked rhS100A11. A unique cross-linked peptide was purified and identified as Met-Ala-Lys3-Ilu-Ser-Ser-Pro-Thr-Glu-Thr-Glu-Arg cross-linked via an Lys3-Gln102 isopeptide bond to Ala-Val-Pro-Ser-Gln102-Lys. These studies show that S100A11 is post-translationally modified by transglutaminase, that it can be cross-linked to form multimers, that it is present in CEs from cultured keratinocytes and in vivo epidermis, and that Lys3 and Gln102 are specific sites of cross-link formation.

Cell Compartmentation↗

PubMed2004

Tissue transglutaminase has intrinsic kinase activity: identification of transglutaminase 2 as an insulin-like growth factor-binding protein-3 kinase.

Tissue transglutaminase (TG2) is a ubiquitous enzyme that cross-links glutamine residues with lysine residues, resulting in protein polymerization, cross-linking of dissimilar proteins, and incorporation of diamines and polyamines into proteins. It has not previously been known to have kinase activity. Recently, insulin-like growth factor-binding protein-3 (IGFBP-3) has been reported to be phosphorylated by breast cancer cell membranes. We purified the IGFBP-3 kinase activity from solubilized T47D breast cancer cell membranes using gel filtration, ion-exchange chromatography, and IGFBP-3 affinity chromatography. The fractions containing kinase activity were further purified by high pressure liquid chromatography and analyzed by tandem mass spectroscopy. TG2 was detected in fractions containing kinase activity. Antisera to TG2 and protein A-Sepharose were used to immunoprecipitate TG2 from membrane fractions. The immunoprecipitates retained IGFBP-3 kinase, whereas immunoprecipitation deleted kinase activity in the membrane supernatant. The inhibitors of TG2, cystamine and monodansyl cadaverine, abolished the ability of the T47D cell membrane preparation to phosphorylate IGFBP-3. Both TG2 purified from guinea pig liver and recombinant human TG2 expressed in insect cells were able to phosphorylate IGFBP-3. TG2 kinase activity was inhibited in a concentration-dependent fashion by calcium, which has previously been shown to be important for the cross-linking activity of TG2. These data provide compelling evidence that TG2 has intrinsic kinase activity, a function that has not previously been ascribed to TG2. Furthermore, we provide evidence that TG2 is a major component of the IGFBP-3 kinase activity present on breast cancer cell membranes.

Animals↗

PubMed2004

Arabidopsis thaliana glutamate-cysteine ligase: functional properties, kinetic mechanism, and regulation of activity.

In plants, glutathione accumulates in response to different stress stimuli as a protective mechanism, but only limited biochemical information is available on the plant enzymes that synthesize glutathione. Glutamatecysteine ligase (GCL) catalyzes the first step in glutathione biosynthesis and plays an important role in regulating the intracellular redox environment. Because the putative Arabidopsis thaliana GCL (AtGCL) displays no significant homology to the GCL from bacteria and other eukaryotes, the identity of this protein as a GCL has been debated. We have purified AtGCL from an Escherichia coli expression system and demonstrated that the recombinant enzyme catalyzes the ATP-dependent formation of gamma-glutamylcysteine from glutamate (Km = 9.1 mm) and cysteine (Km = 2.7 mm). Glutathione feedback inhibits AtGCL (Ki approximately 1.0 mm). As with other GCL, buthionine sulfoximine and cystamine inactivate the Arabidopsis enzyme but with inactivation rates much slower than those of the mammalian, bacterial, and nematode enzymes. The slower inactivation rates observed with AtGCL suggest that the active site differs structurally from that of other GCL. Global fitting analysis of initial velocity data indicates that a random terreactant mechanism with a preferred binding order best describes the kinetic mechanism of AtGCL. Unlike the mammalian GCL, which consists of a catalytic subunit and a regulatory subunit, AtGCL functions and is regulated as a monomeric protein. In response to redox environment, AtGCL undergoes a reversible conformational change that modulates the enzymatic activity of the monomer. These results explain the reported posttranslational change in AtGCL activity in response to oxidative stress.

Adenosine Triphosphate↗

PubMed1978

Biochemical effects on radioprotective agents on the liver microsomal hydroxylating system: in vitro studies.

The action of two radioprotectors--cysteamine and cystamine--on the liver microsomal multi-enzyme hydroxylating system, a key stem in drug and biological compounds metabolism, has been studied. Their effects have been systematically analysed at the level of individual enzyme activities and global functions. The two compounds are quite inactive on NADPH and NADH cytochrome c reductase activities, but slightly denature the cytochrome P450 into cytochrome P420. Furthermore, they do inhibit to some extent (30 per cent at 10(-2) M) the rate of codeine hydroxylation and totally suppress ((at 10(-2) M) the NADPH-induced lipid peroxidation which occurs during enzymatic functioning. These results are discussed in the light of the toxicity of radioprotectors.

Animals↗

PubMed1983

Protection with combinations of hydroxytryptophan and some thiol compounds against whole-body gamma irradiation.

Protection of mice (as measured by survival at 30 days) against whole-body gamma exposure by prior treatment with combinations of hydroxytryptophan (HT) and some thiol drugs, is reported here. The combination of HT and aminoethylisothiuronium bromide hydrobromide (AET) was much more effective than HT alone against the effects of 10.5 and 12.5 Gy of gamma ray exposure. A combination of HT and beta-mercaptopropionylglycine (MPG) gave marked protection against a 10.5 Gy exposure but not against 12.5 Gy. The combination of HT with cysteine or cysteamine also provided reasonable protection against 10.5 Gy. The combination of HT and cystamine rendered a low level of protection against 10.5 Gy, but again it was better than HT alone. The thiol drugs by themselves in the doses used in combination were ineffective in rendering protection after whole body gamma ray exposure.

Animals↗

PubMed1997

Cysteamine oxidation by lentil seedling amine oxidase.

Cysteamine is oxidatively deaminated by lentil amine oxidase. It shows saturation kinetic K(m) = 9 x 10(-4) M like other substrates, but the aldehyde produced leads to loss of enzyme activity, which is restored by dialysis. When putrescine is the substrate of the amine oxidase cysteamine behaves like a competitive inhibitor, and shows Ki = 5 x 10(-5) M. The possible involvement of the oxidation of cysteamine and the inhibitory effects of thioacetaldehyde in the cystamine oxidation by amine oxidase is discussed.

Amine Oxidase (Copper-Containing)↗

PubMed2006

Vanin-1 licenses inflammatory mediator production by gut epithelial cells and controls colitis by antagonizing peroxisome proliferator-activated receptor gamma activity.

Colitis involves immune cell-mediated tissue injuries, but the contribution of epithelial cells remains largely unclear. Vanin-1 is an epithelial ectoenzyme with a pantetheinase activity that provides cysteamine/cystamine to tissue. Using the 2,4,6-trinitrobenzene sulfonic acid (TNBS)-colitis model we show here that Vanin-1 deficiency protects from colitis. This protection is reversible by administration of cystamine or bisphenol A diglycidyl ether, a peroxisome proliferator-activated receptor (PPAR)gamma antagonist. We further demonstrate that Vanin-1, by antagonizing PPARgamma, licenses the production of inflammatory mediators by intestinal epithelial cells. We propose that Vanin-1 is an epithelial sensor of stress that exerts a dominant control over innate immune responses in tissue. Thus, the Vanin-1/pantetheinase activity might be a new target for therapeutic intervention in inflammatory bowel disease.

Active Transport, Cell Nucleus↗

PubMed2003

Transglutaminase potentiates ligand-dependent proteasome dysfunction induced by polyglutamine-expanded androgen receptor.

Expansion of the CAG trinucleotide repeat encoding glutamine in the androgen receptor gene leads to spinobulbar muscular atrophy (SBMA), a neurodegenerative disorder in a family of polyglutamine diseases with enigmatic pathogenic mechanisms. One established property of glutamine residues is their ability to act as an amine accepter in a transglutaminase-catalyzed reaction, resulting in a proteolytically resistant glutamyl-lysine cross-link. To examine underlying disease mechanisms we investigated the relationship between polyglutamine-expanded androgen receptor and transglutaminase. We found androgen receptor N-terminal fragments are a substrate for transglutaminase. Western blots of the proteins following incubation with transglutaminase show that several different epitopes of the AR appear to be lost. We propose that this is due to the transglutaminase cross-linking of the AR, which interferes with antibody recognition. Furthermore, HEK GFP(u)-1 cells expressing polyglutamine-expanded androgen receptor and transglutaminase exhibit ligand-dependent proteasome dysfunction; this effect was not observed in the presence of cystamine, a transglutaminase inhibitor. In addition, transglutaminase-mediated isopeptide bonds were detected in brains of SBMA transgenic mice, but not in controls, suggesting involvement of transglutaminase-catalyzed reactions in polyglutamine disease pathogenesis. Our hypothesis is that cross-linked AR cannot to be degraded by the proteasome and obstructs the proteasome pore, preventing normal function. Because of the central role the ubiquitin-proteasome degradation system plays in fundamental cellular processes, any alteration in its function could cause cell death, ultimately contributing to SBMA pathogenesis.

Animals↗

PubMed2005

Mutant huntingtin protein: a substrate for transglutaminase 1, 2, and 3.

The most prominent neuropathologic hallmarks of Huntington disease (HD) are cortical and striatal perinuclear cytoplasmic aggregates and intranuclear inclusions of mutant huntingtin. Our laboratory previously demonstrated that huntingtin protein colocalizes with transglutaminase 2 and its product, the epsilon-(gamma-glutamyl)lysine bond in intranuclear inclusions in HD frontal cortex. We also found that transglutaminase 2 cross-links N-terminal fragments of mutant huntingtin (htt-N63-148Q-myc) in cells in culture. We now report a significant increase in transglutaminase 2 mRNA in HD cortex (225% of controls) and striatum (399% of controls). Expression of the short transglutaminase 2 mRNA splice variant was not detectable in HD, although previous studies demonstrated upregulation in Alzheimer disease and progressive supranuclear palsy. Cells co-transfected with GFP-tagged transglutaminase 1, 2, or 3 and htt-N63-148Q-myc exhibit increased cross-linked huntingtin in the insoluble fraction of cell lysates. Treatment of cells with cystamine, a chemical inhibitor of transglutaminase, decreased aggregated and cross-linked huntingtin and increased viability of cells that were transfected with transglutaminase 2 and htt-N63-148Q-myc. These data suggest that transglutaminase 1, 2, and 3 could be involved in cross-linking of huntingtin into intranuclear inclusions in HD and that inhibiting transglutaminase should be explored as a potential treatment strategy for HD.

Aged↗

PubMed1983

A versatile method for the coupling of protein to DNA: synthesis of alpha 2-macroglobulin-DNA conjugates.

We describe a simple, general method to link proteins covalently to DNA. The method uses two reagents, N-acetyl-N'-(p-glyoxylylbenzoyl)cystamine and 2-iminothiolane. The former reacts specifically with nonpaired quanine residues and upon reduction generates a free sulfhydryl group. The latter reacts with a protein to provide another sulfhydryl group which is subsequently conjugated to DNA by an intermolecular disulfide interchange reaction. Using this method alpha 2-macroglobulin was conjugated to plasmid DNA encoding the Herpes simplex virus-1 thymidine kinase gene or a DNA fragment containing the E. coli chloramphenicol acetyltransferase gene. Up to 20% of the total DNA was conjugated to alpha 2-macroglobulin and the alpha 2-macroglobulin-DNA conjugate had a protein/DNA molar ratio of approximately two. The whole reaction takes place under very mild conditions in aqueous solution. The structure of DNA appears not to be significantly affected by the chemical modification. This method may prove useful in ligand directed gene transfer studies.

Cross-Linking Reagents↗

PubMed1996

Inhibitory effect of glucose and phosphate on the second cleavage division of hamster embryos: is it linked to metabolism?

Hamster embryos cannot complete the second cleavage division in vitro in the presence of glucose and inorganic phosphate (Glu/Pi). Embryos can recover from an 8 h (12:30-20:30 h) exposure to Glu/Pi, although cleavage is retarded by 10 h and continued development to the blastocyst stage is rare (2%). The vulnerability of 2-cell embryos to Glu/Pi changes during the cell cycle, being greatest just prior to cleavage which occurs at approximately 21:00 h: exposure to Glu/Pi from 12:30-14:30 h allowed 94% development to morulae/blastocysts while exposure from 18:30-20:30 h decreased morula/blastocyst development to 48%. This inhibitory effect is also modulated by the length of exposure and energy substrate composition of the culture medium. It is unclear how Glu/Pi interferes with cleavage, but a metabolic explanation is likely because (i) metabolic inhibitors L-glyceraldehyde, iodoacetate and cystamine exacerbated the effect and (ii) the organization of active mitochondria observed with Rhodamine 123 and confocal microscopy is disturbed by Glu/Pi.

Animals↗

PubMed1978

Formation of interchain disulfide bonds in Bence Jones proteins and Fab(t) fragments of immunoglobulin G through thiol-disulfide interchange.

The formation of interchain disulfide bonds from partially reduced Bence Jones protein (Nag, type lambda) and Fab(t) fragments of IgG1 myeloma proteins was studied in the presence of various disulfide reagents. The results could be well explained in terms of the scheme proposed previously (Kishida et al. (1976) J. Biochem. 79, 91-105). In this scheme, it was assumed that two kinds of intermediate, which form mixed disulfides with either of the paired thiol groups, are produced. For type lambda Bence Jones proteins, only one of the two intermediates can form the inter L-L disulfide bond. The fraction of intermediate having the ability to form the inter L-L disulfide bond was estimated to be 72% of the total Nag protein and was the same irrespective of the kind of disulfide reagent examined. For Fab(t), on the other hand, both intermediates equally can form the inter Fd-L disulfide bond. On the basis of the results with cystamine, it was shown that the formation of an inter Fd-L disulfide bond from the intermediate proceeds about 100 times as rapidly as that of an inter L-L disulfide bond.

Bence Jones Protein↗

PubMed1982

Inhibition by the tetramine disulphide, benextramine, of cardiac chronotropic histamine H2-receptor-mediated effects.

1 Benextramine (N,N1-bis[o-methoxybenzylamino)-n-hexyl]cystamine), which irreversibly blocks alpha-adrenoceptors and does not inhibit the H1-receptor-mediated contractile effect of histamine on guinea-pig isolated ileum, also did not inhibit the H1-receptor-mediated inotropic effect of histamine on guinea-pig isolated atrium. 2 Benextramine irreversibly inhibited the H2-receptor-mediated chronotropic effect of histamine on guinea-pig isolated atrium. 3 Since its combination with the competitive H2-receptor blocking drug cimetidine had a additive blocking effect, benextramine appears to act directly on the chronotropic H2-receptor.

Adrenergic alpha-Antagonists↗

PubMed2004

Vanin-1-/- mice exhibit a glutathione-mediated tissue resistance to oxidative stress.

Vanin-1 is an epithelial ectoenzyme with pantetheinase activity and generating the amino-thiol cysteamine through the metabolism of pantothenic acid (vitamin B(5)). Here we show that Vanin-1(-/-) mice, which lack cysteamine in tissues, exhibit resistance to oxidative injury induced by whole-body gamma-irradiation or paraquat. This protection is correlated with reduced apoptosis and inflammation and is reversed by treating mutant animals with cystamine. The better tolerance of the Vanin-1(-/-) mice is associated with an enhanced gamma-glutamylcysteine synthetase activity in liver, probably due to the absence of cysteamine and leading to elevated stores of glutathione (GSH), the most potent cellular antioxidant. Consequently, Vanin-1(-/-) mice maintain a more reducing environment in tissue after exposure to irradiation. In normal mice, we found a stress-induced biphasic expression of Vanin-1 regulated via antioxidant response elements in its promoter region. This process should finely tune the redox environment and thus change an early inflammatory process into a late tissue repair process. We propose Vanin-1 as a key molecule to regulate the GSH-dependent response to oxidative injury in tissue at the epithelial level. Therefore, Vanin/pantetheinase inhibitors could be useful for treatment of damage due to irradiation and pro-oxidant inducers.

Amidohydrolases↗

PubMed1986

Persulfide sulfur is a growth factor for cells defective in sulfur metabolism.

Several systems which generate persulfide sulfur promote in vitro proliferation of L1210 murine lymphoma cells. The systems include cysteine disulfides and pyridoxal, cystamine and diamine oxidase, beta-mercaptoalcohol disulfides and an alcohol dehydrogenase, and sulfide-treated proteins and a thiol. Persulfide sulfur is very unstable at pH near 7 and an essential feature of the growth-supporting systems is the ability to generate persulfide sulfur at a very low rate for long periods of time. Methyl disulfides (R--S--S--CH3) also support growth of L1210 cells and are more stable than persulfides (R--S--S--H). The requirement for these sulfur groups by L1210 cells may be related to the fact that these cells are defective in at least two enzymes of sulfur metabolism, cystathionase and 5'-methylthioadenosine phosphorylase. These findings provide the first evidence that persulfide sulfur may have a physiological role.

Albumins↗

PubMed1979

Inhibition of the cardiac inotropic effect of phenylephrine by a tetramine disulfide with irreversible alpha-adrenoceptor blocking activity.

The newly synthesized alpha-adrenoceptor blocking drug BHC (N,N'-bis[6-(10-methoxybenzyl-amino)-a-hexyl]cystamine) was found to block irreversibly the positive inotropic effect of the sympathomimetic drug phenylephrine on the isolated rat left atrium. BHC was used to test the adrenoceptor interconversion hypothesis which claims that low temperature converts inotropic beta-adrenoceptors in rat atrium and frog ventricle to alpha-adrenoceptors. There was no evidence of adrenoceptor 'interconversion.' In the rat atrium low temperature did not increase the BHC antagonism of phenylephrine and did not cause BHC to inhibit the inotropic effect of noradrenaline or isoprenaline. In the perfused frog heart low temperature did not lead BHC to inhibit the inotropic effect of phenylephrine, adrenaline, or isoprenaline.

Adrenergic alpha-Antagonists↗

PubMed1980

Potentiation and inhibition of nicotinic effects on striated muscle by the tetramine disulfide benextramine.

In low concentrations (0.3-3 muM) the tetramine disulfide benextramine (BHC; N,N'-bis[6-(o-methoxybenzylamino)-n-hexyl]cystamine) potentiated the contracture of the isolated frog rectus abdominis muscle caused by acetylcholine but in the presence of physostigmine or in a higher concentration (10 muM) it inhibited. Benextramine only inhibited the contracture caused by carbachol or butyrylcholine. The all-carbon analog of benextramine only inhibited the effect of acetylcholine. The inhibitory effects of benextramine and its carbon analog were noncompetitive and readily reversible but the potentiating effect of benextramine was not readily reversible.

Acetylcholine↗

PubMed1978

Alpha-adrenergic receptors in human and canine tracheal and bronchial smooth muscle.

We studied the reactions of human tracheal and bronchial smooth muscle and canine trachealis muscle to adrenergic agonists. Human tissue was obtained from recent autopsies of patients with normal lungs and patients with respiratory disorders, and canine tissue was obtained from animals used in other experiments. The muscle was mounted in tissue baths fitted with platinum wire electrodes and the mechanical activity was recorded. Norepinephrine added to the normal human or canine tissue in concentrations up to 10(-5) M caused no reaction. In the normal human and in the dog, pretreatment with histamine or KCl changed this response and when norepinephrine was added the tissue contracted. This contraction was blocked by phentolamine or N,N'-bis-(O-methoxybenzylaminohexyl)-cystamine tetrahydrochloride (BHC). In diseased tissue, the addition of norepinephrine resulted in a contraction that was blocked by phentolamine or BHC and no pretreatment with histamine or KCl was required. These findings demonstrate alpha-receptors in human and canine airway smooth muscle, and there is a difference between normal and diseased human tissue in the reaction of these tissues to alpha-adrenergic agonists.

Adrenergic alpha-Agonists↗