Intracellular localization and conformational state of transglutaminase 2: implications for cell death.
Intracellular localization and conformational state of transglutaminase 2: implications for cell death.
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DOI：
10.1371/journal.pone.0006123
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发表时间：
2009-07-01
期刊：
影响因子：
3.7
通讯作者：
Johnson GV
中科院分区：
文献类型：
--
作者：
Gundemir S;Johnson GV
转氨酶2（TG 2）是一种多功能酶，除了具有转酰胺功能外，还具有鸟嘌呤核苷酸结合和GTP水解活性。研究表明，TG 2是介导细胞死亡过程的参与者。然而，关于这种酶在细胞死亡过程中的作用还远没有达成共识，因为它似乎取决于细胞类型，刺激，亚细胞定位和酶的构象状态。本研究的目的是剖析TG 2在细胞死亡过程中的作用。为此，我们创建并表征了TG 2的4个不同的点突变体，每个突变体与野生型的不同之处在于其构象或缺乏重要功能。我们还制备了这些突变体作为核靶向蛋白。通过在HEK 293细胞中过表达TG 2的突变体或野生型形式，我们研究了该蛋白在细胞死亡过程中响应于三种应激物的调节作用：毒胡萝卜素、高渗应激和氧/葡萄糖剥夺（OGD）。除了R580 A突变体（其不能结合鸟嘌呤核苷酸，因此更倾向于表现出转酰胺活性）之外，所有的TG 2构建体都没有显著影响细胞死亡过程或者是保护性的。然而，在R580 A突变体的情况下，响应高毒胡萝卜素浓度的细胞死亡显着增加。有趣的是，R580 A-TG 2的核定位足以抵消细胞质R580 A-TG 2的促死亡作用。此外，TG 2的核定位显著促进了其对OGD的保护作用。我们的数据支持这一假设，即转酰胺活性的TG 2，这是大多数静止，除了在极端的压力条件下，是必要的，其促死亡的作用。此外，TG 2的核定位通常在其针对细胞死亡过程的保护功能中起关键作用，或者抵消有害作用或者加强蛋白质的保护作用。
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.
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