Palmitoyl protein thioesterase 1 protects against apoptosis mediated by Ras-Akt-caspase pathway in neuroblastoma cells
Palmitoyl protein thioesterase 1 protects against apoptosis mediated by Ras-Akt-caspase pathway in neuroblastoma cells
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DOI：
10.1046/j.1471-4159.2000.0741478.x
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发表时间：
2000-04-01
影响因子：
4.7
通讯作者：
Dawson, G
中科院分区：
文献类型：
--
作者：
Cho, SG;Dawson, G
棕榈酰蛋白硫酯酶（PPT）1是一种参与棕榈酰化蛋白脱酰的酶。PPT 1的缺乏导致遗传性疾病，婴儿神经元蜡样质脂褐质沉积症，与皮质神经元的大量死亡有关。在过表达PPT 1的人神经母细胞瘤（LA-N-5）细胞中研究了PPT 1在神经元存活和凋亡中的作用。通过脱棕榈酰化活性比基础水平增加200-350%（如通过体外PPT测定所确定的）和通过瞬时表达的表位标记的PPT 1的蛋白质印迹分析两者来显示PPT 1的过表达。过表达的PPT 1表现出相同的酸性pH最佳值（pH 4.0）的内源性酶，当用P-0衍生的八肽底物进行测定，并降低了30%的生长速率。当用C-2-神经酰胺或磷脂酰肌醇3-激酶抑制剂（LY 294002）攻击时，LA-N-5细胞发生凋亡，表现为caspase 3样活性增加和DNA片段化增加。PPT 1的过表达抑制了这种C-2-神经酰胺或LY 294002介导的半胱天冬酶-3活化50%。DNA断裂和细胞死亡也随之减少。与抗凋亡性增加一致，我们发现在PPT 1过表达细胞中抗凋亡蛋白Akt（蛋白激酶B）的磷酸化增加。已知p21（Ras）是动态棕榈酰化和脱棕榈酰化的，并且参与生长和细胞死亡。与对照组相比，C-2-神经酰胺诱导的P21（Ras）的膜结合在PPT 1过表达细胞中减少了30-50%。PPT过表达还导致另一种棕榈酰化蛋白GAP-43（一种神经元特异性蛋白）的膜结合减少。我们的研究表明，蛋白质棕榈酰化可能是细胞凋亡的生理调节因子。
Palmitoyl protein thioesterase (PPT) 1 is an enzyme involved in deacylation of palmitoylated proteins. A deficiency in PPT1 results in a genetic disease, infantile neuronal ceroid lipofuscinosis, associated with massive death of cortical neurons. The role of PPT1 in neuronal survival and apoptosis was studied in human neuroblastoma (LA-N-5) cells overexpressing PPT1. Overexpression of PPT1 was shown both by the 200-350% increase in depalmitoylating activity over basal level (as determined by an in vitro PPT assay) and by western blot analysis of transiently expressed epitope-tagged PPT1. Overexpressed PPT1 showed the same acidic pH optimum (pH 4.0) as the endogenous enzyme, when assayed with a P-0-derived octapeptide substrate, and reduced the growth rate by 30%. LA-N-5 cells underwent apoptosis, as evidenced by increased caspase 3-like activity and increased DNA fragmentation, when challenged with either C-2-ceramide or a phosphatidylinositol 3-kinase inhibitor (LY294002). Overexpression of PPT1 inhibited this C-2-ceramide- or LY294002-mediated activation of caspase-3 by 50%. There was also a concomitant decrease in DNA fragmentation and cell death. Consistent with increased resistance to apoptosis, we found increased phosphorylation of the antiapoptotic protein Akt (protein kinase B) in PPT1-overexpressing cells. p21(Ras) is known to be dynamically palmitoylated and depalmitoylated and is involved in both growth and cell death. The C-2-ceramide-induced membrane association of p21(Ras) was reduced by 30-50% in PPT1-overexpressing cells compared with control. PPT overexpression also led to reduced membrane association of another palmitoylated protein, GAP-43, a neuron-specific protein. Our studies suggest that protein palmitoylation could be a physiological regulator of apoptosis.