Decreased protein S-palmitoylation in dorsolateral prefrontal cortex in schizophrenia.

Recent reports suggest abnormalities of neurotransmitter receptor trafficking, targeting, dendritic localization, recycling, and degradation in the brain in schizophrenia. We hypothesized that a potential explanation for these findings may be abnormal posttranslational modifications that influence intracellular targeting and trafficking of proteins between subcellular compartments. Dysregulation of protein palmitoylation is a strong candidate for such a process. S-palmitoylation is a reversible thioesterification of palmitoyl-groups to cysteine residues that can regulate trafficking and targeting of intracellular proteins. Using a biotin switch assay to study S-palmitoylation of proteins in human postmortem brain, we identified a pattern of palmitoylated proteins that cluster into 17 bands of discrete molecular masses, including numerous proteins associated with receptor signal transduction. Using mass spectrometry, we identified 219 palmitoylated proteins in human frontal cortex, and individually validated palmitoylation status of a subset of these proteins. Next, we assayed protein palmitoylation in dorsolateral prefrontal cortex from 16 schizophrenia patients and paired comparison subjects. S-palmitoylation was significantly reduced for proteins in most of the 17 schizophrenia bands. In rats chronically treated with haloperidol, the same pattern of palmitoylation was observed but the extent of palmitoylation was unchanged, suggesting that the diminution in protein palmitoylation in schizophrenia is not due to chronic antipsychotic treatment. These results indicate there are changes in the extent of S-palmitoylation of many proteins in the frontal cortex in schizophrenia. Given the roles of this posttranslational modification, these data suggest a potential mechanism reconciling previous observations of abnormal intracellular targeting and trafficking of neurotransmitter receptors in this illness.

Tissue transglutaminase overexpression does not modify the disease phenotype of the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) is a devastating autosomal-dominant neurodegenerative disorder initiated by an abnormally expanded polyglutamine in the huntingtin protein. Determining the contribution of specific factors to the pathogenesis of HD should provide rational targets for therapeutic intervention. One suggested contributor is the type 2 transglutaminase (TG2), a multifunctional calcium dependent enzyme. A role for TG2 in HD has been suggested because a polypeptide-bound glutamine is a rate-limiting factor for a TG2-catalyzed reaction, and TG2 can cross-link mutant huntingtin in vitro. Further, TG2 is up regulated in brain areas affected in HD. The objective of this study was to further examine the contribution of TG2 as a potential modifier of HD pathogenesis and its validity as a therapeutic target in HD. In particular our goal was to determine whether an increase in TG2 level, as documented in human HD brains, modulates the well-characterized phenotype of the R6/2 HD mouse model. To accomplish this objective a genetic cross was performed between R6/2 mice and an established transgenic mouse line that constitutively expresses human TG2 (hTG2) under control of the prion promoter. Constitutive expression of hTG2 did not affect the onset and progression of the behavioral and neuropathological HD phenotype of R6/2 mice. We found no alterations in body weight changes, rotarod performances, grip strength, overall activity, and no significant effect on the neuropathological features of R6/2 mice. Overall the results of this study suggest that an increase in hTG2 expression does not significantly modify the pathology of HD.

TG2 protects neuroblastoma cells against DNA-damage-induced stress, suppresses p53 activation.

Tissue transglutaminase (TG2) is a multifunctional member of the transglutaminase (TGase) family (E.C.2.3.2.13), which catalyzes in a calcium-dependent reaction the formation of covalent bonds between the gamma-carboxamide groups of peptide-bound glutamine residues and various primary amines. Here, we investigated the role of TG2 in a response of the neuroblastoma SH-SY5Y cells to topoisomerase II inhibitor etoposide, known to trigger DNA-damage cell response. We found an early and transient (approximately 2 h) increase of the TG2 protein in SH-SY5Y cells treated with etoposide, along with the increase of phosphorylated and total levels of the p53 protein. Next, we showed that SH-SY5Y cells, which overexpress wild-type TG2 were significantly protected against etoposide-induced cell death. The TG2 protective effect was associated only with the transamidation active form of TG2, because overexpression the wild-type TG2, but not its transamidation inactive C277S form, resulted in a pronounced suppression of caspase-3 activity as well as p53 phosphorylation during the etoposide-induced stress. In addition, exacerbation of cell death with a significant increase in caspase-3 and p53 activation was observed in SH/anti-TG2 cells, in which expression of the endogenous TG2 protein has been greatly reduced by the antisense cDNA construct. Though the cell signaling and molecular mechanisms of the TG2-driven suppression of the cell death machinery remain to be investigated, our findings strongly suggest that TG2 plays an active role in the response of neuroblastoma cells to DNA-damage-induced stress by exerting a strong protective effect, likely by the suppression of p53 activation and p53-driven cell signaling events.

Intracellular localization and activity state of tissue transglutaminase differentially impacts cell death.

Tissue transglutaminase (tTG) is a unique member of the transglutaminase family as it is both a transamidating enzyme and a GTPase. In the cell tTG is mostly cytosolic, however it is also found in the nucleus and associated with the plasma membrane. tTG can be proapoptotic, however anti-apoptotic activities of the enzyme have also been reported. To determine how the intracellular localization and transamidating activity of tTG modulates its effects on apoptosis, HEK293 cells were transiently transfected with tTG or [C277S]tTG (which lacks transamidating activity) constructs that were targeted to different intracellular compartments. Apoptosis was induced by thapsigargin treatment, which results in increased intracellular calcium concentrations. Cytosolic tTG was pro-apoptotic, while nuclear localization of [C277S]tTG attenuated apoptosis. Membrane-targeted tTG had neither pro- nor anti-apoptotic functions. This finding indicates for the first time that intracellular localization is an important determinant of the effect of tTG on apoptosis. Previous studies have suggested that tTG may modulate retinoblastoma (Rb) protein, an important suppressor of apoptosis. tTG interacted with Rb and after induction of apoptosis, the interaction of nuclear-targeted [C277S]tTG with Rb was increased significantly concomitant with an attenuation of apoptosis. In contrast, the interaction of nuclear-targeted tTG with Rb was significantly decreased and apoptosis was not attenuated. These data suggest that tTG protects cells against apoptosis in response to stimuli that do not result in increased transamidating activity by translocating to the nucleus, and that complexing with Rb may be an important aspect of the protective effects of tTG.

Tissue transglutaminase directly regulates adenylyl cyclase resulting in enhanced cAMP-response element-binding protein (CREB) activation.

Tissue transglutaminase (tTG) is present in the human nervous system and is predominantly localized to neurons. Treatment of human neuroblastoma SH-SY5Y cells with retinoic acid results in increased tTG expression, which is both necessary and sufficient for differentiation. The goal of the present study was to determine whether tTG modulates the activation of the cyclic AMP-response element (CRE)-binding protein, CREB, an event that likely plays a central role in the differentiation of SH-SY5Y cells. SH-SY5Y cells stably transfected with active wild type tTG, tTG without transamidating activity (C277S), an antisense tTG construct that depleted the endogenous levels of tTG, or vector only were used for the study. Treatment with forskolin, an adenylyl cyclase activator, increased that activation-associated phosphorylation of CREB, which was prolonged by tTG overexpression. CRE-reporter gene activity was also significantly elevated in the tTG cells compared with the other cells. The enhancement of CREB phosphorylation/activation in the tTG cells is likely due to the fact that tTG significantly potentiates cAMP production, and our findings indicate that tTG enhances adenylyl cyclase activity by modulating the conformation state of adenylyl cyclase. This is the first study to provide evidence of the mechanism by which tTG may contribute to neuronal differentiation.

Cystamine inhibits caspase activity. Implications for the treatment of polyglutamine disorders.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an abnormally expended polyglutamine domain. There is no effective treatment for HD; however, inhibition of caspase activity or prevention of mitochondria dysfunction delays disease progression in HD mouse models. Similarly administration of cystamine, which can inhibit transglutaminase, prolonged survival of HD mice, suggesting that inhibition of transglutaminase might provide a new treatment strategy. However, it has been suggested that cystamine may inhibit other thiol-dependent enzymes in addition to transglutaminase. In this study we show that cystamine inhibits recombinant active caspase-3 in a concentration-dependent manner. At low concentrations cystamine is an uncompetitive inhibitor of caspase-3 activity, becoming a non-competitive inhibitor at higher concentrations. The IC(50) for cystamine-mediated inhibition of caspase-3 activity in vitro was 23.6 microm. In situ cystamine inhibited in a concentration-dependent manner the activation of caspase-3 by different pro-apoptotic agents. Additionally, cystamine inhibited caspase-3 activity to the same extent in cell lines stably overexpressing wild type tissue transglutaminase (tTG), a mutant inactive tTG, or an antisense for tTG, demonstrating that cystamine inhibits caspase activity independently of any effects it may have on the transamidating activity of tTG. Finally, treatment with cystamine resulted in a robust increase in the levels of glutathione. These findings demonstrate that cystamine may prolong neuronal survival and delay the onset of HD by inhibiting caspases and increasing the level of antioxidants such as glutathione.

Tissue transglutaminase differentially modulates apoptosis in a stimuli-dependent manner.

Tissue transglutaminase is a unique member of the transglutaminase family as it not only catalyzes a transamidating reaction, but also binds and hydrolyzes GTP and ATP. Tissue transglutaminase has been reported to be pro-apoptotic, however, conclusive evidence is still lacking. To elucidate the role of tissue transglutaminase in the apoptotic process human neuroblastoma SH-SY5Y cells were stably transfected with vector only (SH/pcDNA), wild-type tissue transglutaminase (SH/tTG) and tissue transglutaminase that has no transamidating activity but retains its other functions (SH/C277S). In these studies three different apoptotic stimuli were used osmotic stress, staurosporine treatment and heat shock to delineate the role of tissue transglutaminase as a transamidating enzyme in the apoptotic process. In SH/tTG cells, osmotic stress and staurosporine treatments resulted in significantly greater caspase-3 activation and apoptotic nuclear changes then in SH/pcDNA or SH/C277S cells. This potentiation of apoptosis in SH/tTG cells was concomitant with a significant increase in the in situ transamidating activity of tissue transglutaminase. However, in the heat shock paradigm, which did not result in any increase in the transamidating activity in SH/tTG cells, there was a significant attenuation of caspase-3 activity, LDH release and apoptotic chromatin condensation in SH/tTG and SH/C277S cells compared with SH/pcDNA cells. These findings indicate for the first time that the effect of tissue transglutaminase on the apoptotic process is highly dependent on the type of the stimuli and how the transamidating activity of the enzyme is affected. Tissue transglutaminase facilitates apoptosis in response to stressors that result in an increase in the transamidating activity of the enzyme. However, when the stressors do not result in an increase in the transamidating activity of tissue transglutaminase, than tissue transglutaminase can ameliorate the apoptotic response through a mechanism that is independent of its transamidating function. Further, neither the phosphatidylinositol-3-kinase pathway nor the extracellular-regulated kinase pathway is downstream of the modulatory effects of wild-type tissue transglutaminase or C277S-tissue transglutaminase in the apoptotic cascade.

Does tissue transglutaminase play a role in Huntington's disease?

Tissue transglutaminase (tTG) likely plays a role in numerous processes in the nervous system. tTG posttranslationally modifies proteins by transamidation of specific polypeptide bound glutamines (Glns). This reaction results in the incorporation of polyamines into substrate proteins or the formation of protein crosslinks, modifications that likely have significant effects on neural function. Huntington's disease is a genetic disorder caused by an expansion of the polyglutamine domain in the huntingtin protein. Because a polypeptide bound Gln is the determining factor for a tTG substrate, and mutant huntingtin aggregates have been found in Huntington's disease brain, it has been hypothesized that tTG may contribute to the pathogenesis of Huntington's disease. In vitro, polyglutamine constructs and huntingtin are substrates of tTG. Further, the levels of tTG and TG activity are elevated in Huntington's disease brain and immunohistochemical studies have demonstrated that there is an increase in tTG reactivity in affected neurons in Huntington's disease. These findings suggest that tTG may play a role in Huntington's disease. However in situ, neither wild type nor mutant huntingtin is modified by tTG. Further, immunocytochemical analysis revealed that tTG is totally excluded from the huntingtin aggregates, and modulation of the expression level of tTG had no effect on the frequency of the aggregates in the cells. Therefore, tTG is not required for the formation of huntingtin aggregates, and likely does not play a role in this process in Huntington's disease brain. However, tTG interacts with truncated huntingtin, and selectively polyaminates proteins that are associated with mutant truncated huntingtin. Given the fact that the levels of polyamines in cells is in the millimolar range and the crosslinking and polyaminating reactions catalyzed by tTG are competing reactions, intracellularly polyamination is likely to be the predominant reaction. Polyamination of proteins is likely to effect their function, and therefore it can be hypothesized that tTG may play a role in the pathogenesis of Huntington's disease by modifying specific proteins and altering their function and/or localization. Further research is required to define the specific role of tTG in Huntington's disease.