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Importance of Ca(2+)-dependent transamidation activity in the protection afforded by tissue transglutaminase against doxorubicin-induced apoptosis.

ABSTRACT: Tissue transglutaminase II (TGase-II), which is capable of both GTP binding and transamidation activities, has been implicated in a variety of biological disorders ranging from cancer to neurodegenerative diseases. Recent studies have suggested that the transamidation activity of TGase-II is necessary for the survival of cancer cells confronted with different stresses and cellular insults. When assayed in vitro, the transamidation activity of TGase-II is Ca(2+)-dependent. However, at present, little is known with regard to how the regulation by Ca(2+) is manifested or if in fact it is important for the cellular functions of TGase-II. Here, we have set out to further examine the Ca(2+)-mediated regulation of TGase-II's transamidation activity, with our goals being to identify the Ca(2+)-regulatory sites on the protein and determine whether they are essential for TGase-II to confer survival to human breast cancer cells. On the basis of comparisons between the X-ray crystal structures of TGase-II and TGase-III, we identified three putative Ca(2+)-regulatory sites on TGase-II. Site-directed mutagenesis was performed to individually alter key residues at each of the sites. These substitutions did not affect the ability of TGase-II to bind guanine nucleotides, nor did they cause any obvious changes in its cellular localization. While substitutions at the different Ca(2+)-regulatory sites could either slightly enhance or markedly reduce the GTP hydrolytic activity of TGase-II, mutations at each of the three sites inhibited the Ca(2+)-responsive transamidation activity. We further showed that the same substitutions inhibited the ability of TGase-II to protect human breast cancer cells against the apoptotic activity of doxorubicin. Overall, these findings demonstrate that the Ca(2+)-mediated regulation of transamidation activity is essential for the ability of TGase-II to confer cell survival.

SUBMITTER: Datta S

PROVIDER: S-EPMC2596610 | biostudies-literature | 2006 Nov

REPOSITORIES: biostudies-literature

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Importance of Ca(2+)-dependent transamidation activity in the protection afforded by tissue transglutaminase against doxorubicin-induced apoptosis.

Datta Sunando S Antonyak Marc A MA Cerione Richard A RA

Biochemistry 20061101 44

Tissue transglutaminase II (TGase-II), which is capable of both GTP binding and transamidation activities, has been implicated in a variety of biological disorders ranging from cancer to neurodegenerative diseases. Recent studies have suggested that the transamidation activity of TGase-II is necessary for the survival of cancer cells confronted with different stresses and cellular insults. When assayed in vitro, the transamidation activity of TGase-II is Ca(2+)-dependent. However, at present, li ...[more]

PMID: 17073438

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Project description:BACKGROUND:Outdoor and early evening mosquito biting needs to be addressed if malaria elimination is to be achieved. While indoor-targeted interventions, such as insecticide-treated nets and indoor residual spraying, remain essential, complementary approaches that tackle persisting outdoor transmission are urgently required to maximize the impact. Major malaria vectors principally bite human hosts around the feet and ankles. Consequently, this study investigated whether sandals treated with efficacious spatial repellents can protect against outdoor biting mosquitoes. METHODOLOGY:Sandals affixed with hessian bands measuring 48 cm2 treated with 0.06 g, 0.10 g and 0.15 g of transfluthrin were tested in large cage semi-field and full field experiments. Sandals affixed with hessian bands measuring 240 cm2 and treated with 0.10 g and 0.15 g of transfluthrin were also tested semi field experiments. Human landing catches (HLC) were used to assess reduction in biting exposure by comparing proportions of mosquitoes landing on volunteers wearing treated and untreated sandals. Sandals were tested against insectary reared Anopheles arabiensis mosquitoes in semi-field experiments and against wild mosquito species in rural Tanzania. RESULTS:In semi-field tests, sandals fitted with hessian bands measuring 48 cm2 and treated with 0.15 g, 0.10 g and 0.06 g transfluthrin reduced mosquito landings by 45.9%, (95% confidence interval (C.I.) 28-59%), 61.1% (48-71%), and 25.9% (9-40%), respectively compared to untreated sandals. Sandals fitted with hessian bands measuring 240 cm2 and treated with 0.15 g and 0.10 g transfluthrin reduced mosquito landings by 59% (43-71%) and 64% (48-74%), respectively. In field experiments, sandals fitted with hessian bands measuring 48 cm2 and treated with 0.15 g transfluthrin reduced mosquito landings by 70% (60-76%) against Anopheles gambiae sensu lato, and 66.0% (59-71%) against all mosquito species combined. CONCLUSION:Transfluthrin-treated sandals conferred significant protection against mosquito bites in semi-field and field settings. Further evaluation is recommended for this tool as a potential complementary intervention against malaria. This intervention could be particularly useful for protecting against outdoor exposure to mosquito bites. Additional studies are necessary to optimize treatment techniques and substrates, establish safety profiles and determine epidemiological impact in different settings.

Project description:BackgroundTransglutaminases (TGases) form a group of enzymes that have many different substrates and among the most well known are fibrin for Factor XIIIa and the clotting protein in crustaceans. We also found that TGase is an abundant protein in the hematopoietic tissue (Hpt) cells of crayfish and hence we have studied the possible function of this enzyme in hematopoiesis.ResultsTGase is one of the most abundant proteins in the Hpt and its mRNA expression as well as enzyme activity is very high in the Hpt cells, lesser in the semi-granular hemocytes and very low in the granular cells. In cultured hematopoietic tissues, high activity was present in cells in the centre of the tissue, whereas cells migrating out of the tissue had very low TGase activity. RNAi experiments using dsRNA for TGase completely knocked down the transcript and as a result the cell morphology was changed and the cells started to spread intensely. If astakine, a cytokine directly involved in hematopoiesis, was added the cells started to spread and adopt a morphology similar to that observed after RNAi of TGase. Astakine had no effect on TGase expression, but after a prolonged incubation for one week with this invertebrate cytokine, TGase activity inside and outside the cells was completely lost. Thus it seems as if astakine addition to the Hpt cells and RNAi of TGase in the cell culture will lead to the same results, i.e. loss of TGase activity in the cells and they start to differentiate and spread.ConclusionThe results of this study suggest that TGase is important for keeping the Hpt cells in an undifferentiated stage inside the hematopoietic tissue and if expression of TGase mRNA is blocked the cells start to differentiate and spread. This shows a new function for transglutaminase in preventing hematopoietic stem cells from starting to differentiate and migrate into the hemolymph, whereas their proliferation is unaffected. Astakine is also important for the hematopoiesis, since it induces hemocyte synthesis in the Hpt but now we also show that it in some unknown way participates in the differentiation of the Hpt cells.

Dataset Information

Importance of Ca(2+)-dependent transamidation activity in the protection afforded by tissue transglutaminase against doxorubicin-induced apoptosis.

Publications

Importance of Ca(2+)-dependent transamidation activity in the protection afforded by tissue transglutaminase against doxorubicin-induced apoptosis.

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