Calcium regulates S-nitrosylation, denitrosylation, and activity of tissue transglutaminase
Calcium regulates S-nitrosylation, denitrosylation, and activity of tissue transglutaminase
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DOI：
10.1021/bi002321t
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发表时间：
2001-04-24
期刊：
影响因子：
2.9
通讯作者：
Greenberg, CS
中科院分区：
文献类型：
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作者：
Lai, TS;Hausladen, A;Greenberg, CS
一氧化氮(NO)及其相关分子在血管生物学中发挥着重要作用。NO通过游离半胱氨酸残基的亚硝化来修饰蛋白质，这种修饰在介导NO的生物活性方面具有重要意义。组织转谷氨酰胺酶(TTG)是一种由内皮细胞表达并分泌到细胞外基质(ECM)中的富含巯基的蛋白质，在ECM中它与纤维连接蛋白结合。组织TG具有依赖于钙离子的谷氨酰胺转氨酶活性(TGase)，该活性使参与伤口愈合、组织重塑和细胞外基质稳定的蛋白质发生交叉连接。由于tTG靠近NO产生部位，有18个游离半胱氨酸残基，并利用半胱氨酸进行催化，因此我们研究了调节NO结合和tTG活性的因素。我们报道了TGase活性受NO通过一种独特的钙依赖机制调节。组织甘油三酯可以被一氧化氮载体S亚硝基半胱氨酸(CysNO)聚合为S亚硝基。在没有钙离子的情况下，多达8个半胱氨酸在不改变TGase活性的情况下被亚硝化。在有钙离子存在的情况下，发现多达15个半胱氨酸被硝化，这种修饰导致了TGase活性的抑制。在硝酸化的tTG中加入Ca~(2+)能够触发NO基团的释放(即反硝化)。在无钙条件下，TTG亚硝化反应对镁-GTP的抑制作用增加6倍。当培养的内皮细胞与TTG共同孵育并刺激产生NO时，外源性TTG被S亚硝化。此外，S-硝酸甘油酯还能抑制二磷酸腺苷诱导的血小板聚集。综上所述，我们提供了钙离子调节甘油三酯的S-亚硝化和脱亚硝化从而调节甘氨酸甘油酶活性的证据。这些结果表明，Ca~(2+)在调节NO对tTG的抑制中具有新的变构作用，而tTG在分配NO生物活性中具有新的功能。
Nitric oxide (NO) and related molecules play important roles in vascular biology. NO modifies proteins through nitrosylation of free cysteine residues, and such modifications are important in mediating NO's biologic activity. Tissue transglutaminase (tTG) is a sulfhydryl rich protein that is expressed by endothelial cells and secreted into the extracellular matrix (ECM) where it is bound to fibronectin. Tissue TG exhibits a Ca2+-dependent transglutaminase activity (TGase) that cross-links proteins involved in wound healing, tissue remodeling, and ECM stabilization. Since tTG is in proximity to sites of NO production, has 18 free cysteine residues, and utilizes a cysteine for catalysis, we investigated the factors that regulated NO binding and tTG activity. We report that TGase activity is regulated by NO through a unique Ca2+-dependent mechanism. Tissue TG can be poly-S-nitrosylated by the NO carrier, S-nitrosocysteine (CysNO). In the absence of Ca2+, up to eight cysteines were nitrosylated without modifying TGase activity. In the presence of Ca2+, up to 15 cysteines were found to be nitrosylated and this modification resulted in an inhibition of TGase activity. The addition of Ca2+ to nitrosylated tTG was able to trigger the release of NO groups (i.e. denitrosylation). tTG nitrosylated in the absence of Ca2+ was 6-fold more susceptible to inhibition by Mg-GTP. When endothelial cells in culture were incubated with tTG and stimulated to produce NO, the exogenous tTG was S-nitrosylated. Furthermore, S-nitrosylated tTG inhibited platelet aggregation induced by ADP. In conclusion, we provide evidence that Ca2+ regulates the S-nitrosylation and denitrosylation of tTG and thereby TGase activity. These data suggest a novel allosteric role for Ca2+ in regulating the inhibition of tTG by NO and a novel function for tTG in dispensing NO bioactivity.