Nakajima Hidemitsu, Itakura Masanori, Kubo Takeya, Kaneshige Akihiro, Harada Naoki, Izawa Takeshi, Azuma Yasu-Taka, Kuwamura Mitsuru, Yamaji Ryouichi, Takeuchi Tadayoshi
From the Laboratory of Veterinary Pharmacology,
From the Laboratory of Veterinary Pharmacology.
J Biol Chem. 2017 Mar 17;292(11):4727-4742. doi: 10.1074/jbc.M116.759084. Epub 2017 Feb 6.
Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein that also mediates cell death under oxidative stress. We reported previously that the active-site cysteine (Cys-152) of GAPDH plays an essential role in oxidative stress-induced aggregation of GAPDH associated with cell death, and a C152A-GAPDH mutant rescues nitric oxide (NO)-induced cell death by interfering with the aggregation of wild type (WT)-GAPDH. However, the detailed mechanism underlying GAPDH aggregate-induced cell death remains elusive. Here we report that NO-induced GAPDH aggregation specifically causes mitochondrial dysfunction. First, we observed a correlation between NO-induced GAPDH aggregation and mitochondrial dysfunction, when GAPDH aggregation occurred at mitochondria in SH-SY5Y cells. In isolated mitochondria, aggregates of WT-GAPDH directly induced mitochondrial swelling and depolarization, whereas mixtures containing aggregates of C152A-GAPDH reduced mitochondrial dysfunction. Additionally, treatment with cyclosporin A improved WT-GAPDH aggregate-induced swelling and depolarization. In doxycycline-inducible SH-SY5Y cells, overexpression of WT-GAPDH augmented NO-induced mitochondrial dysfunction and increased mitochondrial GAPDH aggregation, whereas induced overexpression of C152A-GAPDH significantly suppressed mitochondrial impairment. Further, NO-induced cytochrome release into the cytosol and nuclear translocation of apoptosis-inducing factor from mitochondria were both augmented in cells overexpressing WT-GAPDH but ameliorated in C152A-GAPDH-overexpressing cells. Interestingly, GAPDH aggregates induced necrotic cell death via a permeability transition pore (PTP) opening. The expression of either WT- or C152A-GAPDH did not affect other cell death pathways associated with protein aggregation, such as proteasome inhibition, gene expression induced by endoplasmic reticulum stress, or autophagy. Collectively, these results suggest that NO-induced GAPDH aggregation specifically induces mitochondrial dysfunction via PTP opening, leading to cell death.
糖酵解途径中的甘油醛-3-磷酸脱氢酶（GAPDH）是一种多功能蛋白，在氧化应激条件下也介导细胞死亡。我们之前报道过，GAPDH的活性位点半胱氨酸（Cys-152）在氧化应激诱导的与细胞死亡相关的GAPDH聚集过程中起关键作用，并且C152A-GAPDH突变体通过干扰野生型（WT）-GAPDH的聚集来挽救一氧化氮（NO）诱导的细胞死亡。然而，GAPDH聚集体诱导细胞死亡的详细机制仍不清楚。在此我们报道，NO诱导的GAPDH聚集特异性地导致线粒体功能障碍。首先，当GAPDH聚集在SH-SY5Y细胞的线粒体中发生时，我们观察到NO诱导的GAPDH聚集与线粒体功能障碍之间存在相关性。在分离的线粒体中，WT-GAPDH聚集体直接诱导线粒体肿胀和去极化，而含有C152A-GAPDH聚集体的混合物可减轻线粒体功能障碍。此外，用环孢素A处理可改善WT-GAPDH聚集体诱导的肿胀和去极化。在强力霉素诱导的SH-SY5Y细胞中，WT-GAPDH的过表达增强了NO诱导的线粒体功能障碍并增加了线粒体GAPDH聚集，而诱导过表达C152A-GAPDH则显著抑制了线粒体损伤。此外，在过表达WT-GAPDH的细胞中，NO诱导的细胞色素从线粒体释放到细胞质以及凋亡诱导因子从线粒体的核转位均增强，但在过表达C152A-GAPDH的细胞中则得到改善。有趣的是，GAPDH聚集体通过通透性转换孔（PTP）开放诱导坏死性细胞死亡。WT-或C152A-GAPDH的表达均不影响与蛋白质聚集相关的其他细胞死亡途径，如蛋白酶体抑制、内质网应激诱导的基因表达或自噬。总的来说，这些结果表明，NO诱导的GAPDH聚集通过PTP开放特异性地诱导线粒体功能障碍，从而导致细胞死亡。
