The complex role of transglutaminase 2 in glioblastoma proliferation.

BACKGROUND: Glioblastomas (GBMs) are a heterogeneous group of primary brain tumors. These tumors are resistant to therapeutic interventions and invariably recur after surgical resection. The multifunctional protein transglutaminase 2 (TG2) has been shown to promote cell survival in a number of different tumors. There is also evidence that TG2 may be a pro-survival factor in GBMs. However, the roles that TG2 plays in facilitating GBM survival and proliferation have not yet been clearly delineated . METHODS: The functions of TG2 are often cell- and context-specific. Therefore, in this study we examined the ability of TG2 to facilitate GBM proliferation using colony formation assays and 5-ethynyl-2'-deoxyuridine (EdU) incorporation in several different GBM cell lines as well as neurospheres derived from patient tumors representing the 3 major subtypes of GBM tumors (mesenchymal, proneural, and classical) and maintained in the absence of serum. TG2 knockdown or selective TG2 inhibitors were used to modulate TG2 expression and activity. RESULTS: We show that TG2 plays differential roles in the proliferative process depending on the cell type. In most, but not all, GBM models TG2 plays a crucial role in the proliferative process, and some but not all TG2 inhibitors were highly effective at reducing proliferation in a large subset of the GBM models. CONCLUSION: Our results show that TG2 plays an important-but notoriously context-specific-role in GBM cell biology. Nonetheless, as future studies unravel the genetic "fingerprints" that make TG2 inhibitors effective, this information could be exploited to develop TG2 inhibitors into personalized GBM therapies.

Nrf2 reduces levels of phosphorylated tau protein by inducing autophagy adaptor protein NDP52.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal transcription factor in the defence against oxidative stress. Here we provide evidence that activation of the Nrf2 pathway reduces the levels of phosphorylated tau by induction of an autophagy adaptor protein NDP52 (also known as CALCOCO2) in neurons. The expression of NDP52, which we show has three antioxidant response elements (AREs) in its promoter region, is strongly induced by Nrf2, and its overexpression facilitates clearance of phosphorylated tau in the presence of an autophagy stimulator. In Nrf2-knockout mice, phosphorylated and sarkosyl-insoluble tau accumulates in the brains concurrent with decreased levels of NDP52. Moreover, NDP52 associates with phosphorylated tau from brain cortical samples of Alzheimer disease cases, and the amount of phosphorylated tau in sarkosyl-insoluble fractions is inversely proportional to that of NDP52. These results suggest that NDP52 plays a key role in autophagy-mediated degradation of phosphorylated tau in vivo.

Impaired mitochondrial dynamics and Nrf2 signaling contribute to compromised responses to oxidative stress in striatal cells expressing full-length mutant huntingtin.

Huntington disease (HD) is an inherited neurodegenerative disease resulting from an abnormal expansion of polyglutamine in huntingtin (Htt). Compromised oxidative stress defense systems have emerged as a contributing factor to the pathogenesis of HD. Indeed activation of the Nrf2 pathway, which plays a prominent role in mediating antioxidant responses, has been considered as a therapeutic strategy for the treatment of HD. Given the fact that there is an interrelationship between impairments in mitochondrial dynamics and increased oxidative stress, in this present study we examined the effect of mutant Htt (mHtt) on these two parameters. STHdh(Q111/Q111) cells, striatal cells expressing mHtt, display more fragmented mitochondria compared to STHdh(Q7/Q7) cells, striatal cells expressing wild type Htt, concurrent with alterations in the expression levels of Drp1 and Opa1, key regulators of mitochondrial fission and fusion, respectively. Studies of mitochondrial dynamics using cell fusion and mitochondrial targeted photo-switchable Dendra revealed that mitochondrial fusion is significantly decreased in STHdh(Q111/Q111) cells. Oxidative stress leads to dramatic increases in the number of STHdh(Q111/Q111) cells containing swollen mitochondria, while STHdh(Q7/Q7) cells just show increases in the number of fragmented mitochondria. mHtt expression results in reduced activity of Nrf2, and activation of the Nrf2 pathway by the oxidant tBHQ is significantly impaired in STHdh(Q111/Q111) cells. Nrf2 expression does not differ between the two cell types, but STHdh(Q111/Q111) cells show reduced expression of Keap1 and p62, key modulators of Nrf2 signaling. In addition, STHdh(Q111/Q111) cells exhibit increases in autophagy, whereas the basal level of autophagy activation is low in STHdh(Q7/Q7) cells. These results suggest that mHtt disrupts Nrf2 signaling which contributes to impaired mitochondrial dynamics and may enhance susceptibility to oxidative stress in STHdh(Q111/Q111) cells.

Transglutaminase 2 facilitates or ameliorates HIF signaling and ischemic cell death depending on its conformation and localization.

Transglutaminase 2 (TG2) is a widely expressed and multifunctional protein that modulates cell death/survival processes. We have previously shown that TG2 binds to hypoxia inducible factor 1ß (HIF1ß) and decreases the upregulation of HIF responsive genes; however, the relationship between these observations was not investigated. In this study, we investigated whether endogenous TG2 is sufficient to suppress HIF activity and whether the interaction between TG2 and HIF1ß is required for this suppression. shRNA-mediated silencing of TG2 significantly enhanced HIF activation in response to hypoxia. In addition, nuclear localization of TG2 is required for its suppressive effect on HIF activity, with TG2 being recruited to HIF responsive promoters in hypoxic conditions. These observations suggest that TG2 directly regulates hypoxic transcriptional machinery; however, its interaction with HIF1ß was not required for this regulation. We also examined whether TG2's effect on cell death/survival processes in ischemia is due to its effects on HIF signaling. Our results indicate that TG2 mediated HIF suppression can be separated from TG2's effect on cell survival in hypoxic/hypoglycemic conditions. Lastly, here we show that nuclear TG2 in the closed conformation and non-nuclear TG2 in the open conformation have opposing effects on hypoxic/hypoglycemic cell death, which could explain previous controversial results. Overall, our results further clarify the role of TG2 in mediating the cellular response to ischemia and suggest that manipulating the conformation of TG2 might be of pharmacological interest as a therapeutic strategy for the treatment of ischemia-related pathologies.

Transglutaminase 2: a molecular Swiss army knife.

Transglutaminase 2 (TG2) is the most widely distributed member of the transglutaminase family with almost all cell types in the body expressing TG2 to varying extents. In addition to being widely expressed, TG2 is an extremely versatile protein exhibiting transamidating, protein disulphide isomerase and guanine and adenine nucleotide binding and hydrolyzing activities. TG2 can also act as a protein scaffold or linker. This unique protein also undergoes extreme conformational changes and exhibits localization diversity. Being mainly a cytosolic protein; it is also found in the nucleus, associated with the cell membrane (inner and outer side) and with the mitochondria, and also in the extracellular matrix. These different activities, conformations and localization need to be carefully considered while assessing the role of TG2 in physiological and pathological processes. For example, it is becoming evident that the role of TG2 in cell death processes is dependent upon the cell type, stimuli, subcellular localization and conformational state of the protein. In this review we discuss in depth the conformational and functional diversity of TG2 in the context of its role in numerous cellular processes. In particular, we have highlighted how differential localization, conformation and activities of TG2 may distinctly mediate cell death processes.

Intracellular localization and conformational state of transglutaminase 2: implications for cell death.

Transglutaminase 2 (TG2) is a multifunctional enzyme that has guanine nucleotide binding and GTP hydrolyzing activity in addition to its transamidating function. Studies show that TG2 is a player in mediating cell death processes. However, there is far from a consensus about the role of this enzyme in cell death processes as it appears to be dependent upon the cell type, stimuli, subcellular localization and conformational state of the enzyme. The purpose of this study was to dissect the role of TG2 in the cell death processes. To this end, we created and characterized 4 distinct point mutants of TG2, each of which differs from the wild type by its conformation or by lacking an important function. We also prepared these mutants as nuclear targeted proteins. By overexpressing mutant or wild type forms of TG2 in HEK 293 cells, we investigated the modulatory role of the protein in the cell death process in response to three stressors: thapsigargin, hyperosmotic stress and oxygen/glucose deprivation (OGD). All of the TG2 constructs, except the R580A mutant (which cannot bind guanine nucleotides and is therefore more prone to exhibit transamidating activity), either did not significantly affect the cell death processes or were protective. However in the case of the R580A mutant, cell death in response to high thapsigargin concentrations, was significantly increased. Intriguingly, nuclear localization of R580A-TG2 was sufficient to counteract the pro-death role of cytoplasmic R580A-TG2. In addition, nuclear localization of TG2 significantly facilitated its protective role against OGD. Our data support the hypothesis that the transamidation activity of TG2, which is mostly quiescent except in extreme stress conditions, is necessary for its pro-death role. In addition, nuclear localization of TG2 generally plays a key role in its protective function against cell death processes, either counteracting the detrimental effect or strengthening the protective role of the protein.

The Differential Effects of R580A Mutation on Transamidation and GTP Binding Activity of Rat and Human Type 2 Transglutaminase.

Type 2 transglutaminase (TG2) is an acyltransferase, which also undergoes a GTP-binding/GTPase cycle, with guanine nucleotide and calcium binding reciprocally regulating its transamidation (TG) activity. TG2 is expressed ubiquitously throughout the human body and is the predominant neuronal transglutaminase. Given a postulated role for TG2 in a number of physiological and pathological processes including neurodegenerative diseases, it is of critical importance to understand how TG2 and its enzymatic activities are regulated in the cells. The various aspects of TG2 regulation are addressed by using rat and human TG2 proteins, however, despite their homologous structure, regulation of their enzymatic activities may differ, especially in the cellular context. Here, we evaluate the role of Arg580 in human TG2 and Arg579 in rat TG2 in modulating GTP binding and TG activities in vitro and in situ. We confirm the importance of Arg580 and Arg579 in TG2 for GTP binding as their mutation to Ala completely abolished GTP binding activity in both human (R580A) and rat TG2 (R579A). Next, we showed that in transfected human embryonic kidney (HEK) 293 cells, basal in situ TG activity of human R580A TG2 and rat R579A TG2 was significantly greater than their wild-type (WT) counterparts. However, TG activity of the mutants and WT TG2 became equivalent when the intracellular calcium concentration was maximally increased with maitotoxin. Also, in vitro TG activity assay revealed an intriguing difference between rat and human TG2; at a calcium concentration when their activities were maximum, the protein level of human R580A TG2 was lower than its WT counterpart, whereas rat R579A and WT TG2 protein levels were similar. Taken together, our study underscores an essential role of Arg580 in human TG2 and Arg579 in rat TG2 for their GTP binding ability and also describes for the first time that these amino acid residues differentially influence the TG activity of human or rat TG2 by calcium in vitro and in situ.

Transglutaminase 2 protects against ischemic insult, interacts with HIF1beta, and attenuates HIF1 signaling.

Transglutaminase 2 (TG2) is a multifunctional enzyme that has been implicated in the pathogenesis of neurodegenerative diseases, ischemia, and stroke. The mechanism by which TG2 modulates disease progression have not been elucidated. In this study we investigate the role of TG2 in the cellular response to ischemia and hypoxia. TG2 is up-regulated in neurons exposed to oxygen and glucose deprivation (OGD), and increased TG2 expression protects neurons against OGD-induced cell death independent of its transamidating activity. We identified hypoxia inducible factor 1beta (HIF1beta) as a TG2 binding partner. HIF1beta and HIF1alpha together form the heterodimeric transcription factor hypoxia inducible factor 1 (HIF1). TG2 and the transaminase-inactive mutant C277S-TG2 inhibited a HIF-dependent transcription reporter assay under hypoxic conditions without affecting nuclear protein levels for HIF1alpha or HIF1beta, their ability to form the HIF1 heterodimeric transcription factor, or HIF1 binding to its DNA response element. Interestingly, TG2 attenuates the up-regulation of the HIF-dependent proapoptotic gene Bnip3 in response to OGD but had no effect on the expression of VEGF, which has been linked to prosurvival processes. This study demonstrates for the first time that TG2 protects against OGD, interacts with HIF1beta, and attenuates the HIF1 hypoxic response pathway. These results indicate that TG2 may play an important role in protecting against the delayed neuronal cell death in ischemia and stroke.