Saunders P A, Chen R W, Chuang D M
Section on Molecular Neurobiology, Biological Psychiatry Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892-1272, USA.
J Neurochem. 1999 Mar;72(3):925-32. doi: 10.1046/j.1471-4159.1999.0720925.x.
Treatment with cytosine beta-D-arabinoside (AraC; 300 microM) induced a time-dependent accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in nuclei purified from cultured cerebellar granule cells, with a concomitant degradation of lamin B1, a nuclear membrane protein and a substrate of CPP32/caspase-3. Moreover, Asp-Glu-Val-Asp-fluoromethyl ketone (DEVD-fmk), a CPP32-selective antagonist, dose-dependently suppressed AraC-induced apoptosis of these neurons. Nuclear accumulation of GAPDH protein was associated with a progressive decrease in the activity of uracil-DNA glycosylase (UDG), one of the nuclear functions of GAPDH. The nuclear dehydrogenase activity of GAPDH was initially increased after treatment and then decreased parallel to UDG activity. Six GAPDH isoforms were detected in the nuclei of AraC-treated cells. The more alkaline isoforms, 1-3, constituted the bulk of the nuclear GAPDH, and the remaining isoforms, 4-6, were the minor species. Levels of all six isoforms were increased after treatment with AraC for 16 h; a 4-h treatment increased levels of only isoforms 4 and 5. Thus, it appears that various GAPDH isoforms are differentially regulated and may have distinct apoptotic roles. Pretreatment with GAPDH antisense oligonucleotide blocked the nuclear translocation of GAPDH isoforms, and the latter process occurred concurrently with a decrease in cytosolic GAPDH isoforms. Sodium nitroprusside-induced NAD labeling of nuclear GAPDH showed a 60% loss of GAPDH labeling after AraC treatment, suggesting that the active site of GAPDH may be covalently modified, denatured, or improperly folded. The unfolded protein response elicited by denatured GAPDH may contribute to AraC-induced neuronal death.
用β-D-阿拉伯糖胞苷（AraC；300微摩尔）处理可诱导从小脑颗粒细胞培养物中纯化的细胞核中甘油醛-3-磷酸脱氢酶（GAPDH）蛋白随时间积累，同时核膜蛋白和CPP32/半胱天冬酶-3底物层粘连蛋白B1降解。此外，CPP32选择性拮抗剂天冬氨酸-谷氨酸-缬氨酸-天冬氨酸-氟甲基酮（DEVD-fmk）剂量依赖性地抑制AraC诱导的这些神经元凋亡。GAPDH蛋白的核积累与尿嘧啶-DNA糖基化酶（UDG）活性的逐渐降低相关，UDG是GAPDH的核功能之一。GAPDH的核脱氢酶活性在处理后最初增加，然后与UDG活性平行降低。在AraC处理的细胞的细胞核中检测到六种GAPDH同工型。碱性更强的同工型1-3构成了核GAPDH的大部分，其余同工型4-6是少数种类。用AraC处理16小时后，所有六种同工型的水平均升高；处理4小时仅增加了同工型4和5的水平。因此，似乎各种GAPDH同工型受到不同的调节，可能具有不同的凋亡作用。用GAPDH反义寡核苷酸预处理可阻断GAPDH同工型的核转位，后一过程与胞质GAPDH同工型的减少同时发生。硝普钠诱导的核GAPDH的NAD标记显示，AraC处理后GAPDH标记损失60%，这表明GAPDH的活性位点可能被共价修饰、变性或折叠不当。变性GAPDH引发的未折叠蛋白反应可能导致AraC诱导的神经元死亡。
