Hisamatsu Yosuke, Suzuki Nozomi, Masum Abdullah-Al, Shibuya Ai, Abe Ryo, Sato Akira, Tanuma Sei-Ichi, Aoki Shin
Faculty of Pharmaceutical Sciences, ‡Research Institute for Biomedical Sciences, §Division of Medical-Science-Engineering Cooperation and ∥Imaging Frontier Center, Research Institute for Science and Technology, Tokyo University of Science , 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
Bioconjug Chem. 2017 Feb 15;28(2):507-523. doi: 10.1021/acs.bioconjchem.6b00627. Epub 2016 Dec 29.
In our previous paper, we reported on the preparation of some cationic amphiphilic Ir complexes (2c, 2d) containing KKGG peptides that induce and detect cell death of Jurkat cells. Mechanistic studies suggest that 2c interacts with anionic molecules and/or membrane receptors on the cell surface to trigger an intracellular Ca response, resulting in the induction of cell death, accompanied by membrane disruption. We have continued the studies of cell death of Jurkat cells induced by 2c and found that xestospongin C, a selective inhibitor of an inositol 1,4,5-trisphosphate receptor located on the endoplasmic reticulum (ER), reduces the cytotoxicity of 2c, suggesting that 2c triggers the release of Ca from the ER, leading to an increase in the concentration of cytosolic Ca, thus inducing cell death. Moreover, we synthesized a series of new amphiphilic cationic Ir complexes 5a-c containing photoreactive 3-trifluoromethyl-3-phenyldiazirine (TFPD) groups, in an attempt to identify the target molecules of 2c. Interestingly, it was discovered that a TFPD group functions as a triplet quencher of Ir complexes. It was also found that 5b is useful as a turn-on phosphorescent probe of acidic proteins such as bovine serum albumin (BSA) (pI = 4.7) and their complexation was confirmed by luminescence titrations and SDS-PAGE of photochemical products between them. These successful results allowed us to carry out photoaffinity labeling of the target biomolecules of 5b (2c and analogues thereof) in Jurkat cells. A proteomic analysis of the products obtained by the photoirradiation of 5b with Jurkat cells suggests that the Ca-binding protein "calmodulin (CaM)" is one of target proteins of the Ir complexes. Indeed, 5b was found to interact with the Ca-CaM complex, as evidenced by luminescence titrations and the results of photochemical reactions of 5b with CaM in the presence of Ca (SDS-PAGE). A plausible mechanism for cell death induced by a cationic amphiphilic Ir complex is discussed on the basis of our results.
在我们之前的论文中，我们报道了一些含有KKGG肽的阳离子两亲性铱配合物（2c、2d）的制备，这些配合物可诱导并检测Jurkat细胞的细胞死亡。机理研究表明，2c与细胞表面的阴离子分子和/或膜受体相互作用，触发细胞内钙反应，导致细胞死亡的诱导，并伴有膜破坏。我们继续对2c诱导的Jurkat细胞死亡进行研究，发现位于内质网（ER）上的肌醇1,4,5-三磷酸受体的选择性抑制剂西司他汀C可降低2c的细胞毒性，这表明2c触发了内质网中钙的释放，导致胞质钙浓度增加，从而诱导细胞死亡。此外，我们合成了一系列含有光反应性3-三氟甲基-3-苯基重氮甲烷（TFPD）基团的新型两亲性阳离子铱配合物5a-c，试图确定2c的靶分子。有趣的是，发现TFPD基团可作为铱配合物的三线态猝灭剂。还发现5b可用作牛血清白蛋白（BSA）（pI = 4.7）等酸性蛋白质的开启型磷光探针，并且通过发光滴定和它们之间光化学产物的SDS-PAGE证实了它们的络合。这些成功的结果使我们能够在Jurkat细胞中对5b（2c及其类似物）的靶生物分子进行光亲和标记。对用Jurkat细胞对5b进行光照射所获得产物的蛋白质组学分析表明，钙结合蛋白“钙调蛋白（CaM）”是铱配合物的靶蛋白之一。实际上，通过发光滴定以及5b在钙存在下与CaM的光化学反应结果（SDS-PAGE）证明，5b与Ca-CaM复合物相互作用。基于我们的结果，讨论了阳离子两亲性铱配合物诱导细胞死亡的合理机制。
