Natural Product Research Institute, College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, South Korea.
Natural Product Research Institute, College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, South Korea; Stem Cells and Metabolism Research Program, Faculty of Medicine / Helsinki Institute of Life Science, University of Helsinki, Finland.
Biochem Biophys Res Commun. 2020 Mar 26;524(1):224-230. doi: 10.1016/j.bbrc.2020.01.073. Epub 2020 Jan 23.
Isocitrate dehydrogenase (IDH) mutations are found in low-grade gliomas, and the product of the IDH mutant (MT), 2-hydroxyglutarate (2-HG), is the first known oncometabolite. However, the roles of the IDH wild type (WT) in high-grade glioblastoma, which rarely has the IDH mutation, are still unknown. To investigate possible pathways related to IDH WT in gliomas, we carried out bioinformatics analysis, and found that IDH1 has several putative calmodulin (CaM) binding sites. Pull-down and quantitative dissociation constant (Kd) measurements using recombinant proteins showed that IDH1 WT indeed binds to CaM with a higher affinity than IDH1 R132H MT. This biochemical interaction was demonstrated also in the cellular environment by immunoprecipitation with glioblastoma cell extracts. A synthetic peptide for the suggested binding region interfered with the interaction between CaM and IDH1, confirming the specificity of the binding. Direct binding between the synthetic peptide and CaM was observed in an NMR binding experiment, which additionally revealed that the peptide initially binds to the C-lobe of CaM. The physiological meaning of the CaM-IDH1 WT binding was shown with trifluoperazine (TFP), a CaM antagonist, which disrupted the binding and inhibited survival and migration of glioblastoma cells with IDH1 WT. As CaM signaling is activated in glioblastoma, our results suggest that IDH1 WT may be involved in the CaM-signaling pathway in the tumorigenesis of high-grade gliomas.
异柠檬酸脱氢酶（IDH）突变存在于低级别胶质瘤中，IDH 突变的产物（MT），2-羟基戊二酸（2-HG），是第一个已知的致癌代谢物。然而，在高级别脑胶质瘤中，IDH 野生型（WT）的作用仍然未知，因为这些肿瘤很少发生 IDH 突变。为了研究 IDH WT 在胶质瘤中可能相关的途径，我们进行了生物信息学分析，发现 IDH1 有几个潜在的钙调蛋白（CaM）结合位点。使用重组蛋白进行下拉和定量解离常数（Kd）测量表明，IDH1 WT 确实与 CaM 具有更高的亲和力，而不是 IDH1 R132H MT。这种生化相互作用在胶质瘤细胞提取物的免疫沉淀实验中也在细胞环境中得到了证明。用于结合区域的合成肽干扰了 CaM 和 IDH1 之间的相互作用，证实了结合的特异性。在 NMR 结合实验中观察到合成肽与 CaM 之间的直接结合，此外还表明该肽最初结合到 CaM 的 C 结构域。钙调蛋白拮抗剂三氟拉嗪（TFP）破坏了 IDH1 WT 与 CaM 的结合，并抑制了具有 IDH1 WT 的脑胶质瘤细胞的存活和迁移，从而显示了 CaM-IDH1 WT 结合的生理意义。由于 CaM 信号在脑胶质瘤中被激活，我们的结果表明 IDH1 WT 可能参与了高级别脑胶质瘤发生过程中的 CaM 信号通路。
