Identifying the Potential Substrates of the Depalmitoylation Enzyme Acyl-protein Thioesterase 1
Identifying the Potential Substrates of the Depalmitoylation Enzyme Acyl-protein Thioesterase 1
复制标题
鉴定去棕榈酰化酶酰基蛋白硫酯酶 1 的潜在底物
DOI：
10.2174/1566524019666190325143412
复制
发表时间：
2019-01-01
影响因子：
2.5
通讯作者：
Kong, Eryan
中科院分区：
文献类型：
--
作者：
Liu, Huicong;Yan, Peipei;Kong, Eryan
背景：棕榈酰化与去棕榈酰化的动态平衡参与多种细胞过程，其失衡会引发严重的生理后果。酰基蛋白硫酯酶（APT）和棕榈酰蛋白硫酯酶（PPT）催化去棕榈酰化过程。人类PPT1的自然突变会引发神经退行性疾病，然而对APT1的了解仍有待阐明。虽然小鼠中APT1基因缺失可能导致胚胎致死，但对其功能的解读严格依赖于对其底物的识别。
目的：利用构建的人APT1基因敲除细胞系确定APT1的潜在底物。
方法：采用棕榈酰化蛋白富集与质谱联用技术分析差异蛋白。通过树脂辅助捕获（RAC）法提取人胚肾293T细胞（HEK293T）和APT1基因敲除细胞中的棕榈酰化蛋白，并运用蛋白质组学数据非依赖采集（DIA）定量方法进行数据收集。
结果：共鉴定出382种蛋白质。基因本体分类将这些蛋白质划分到不同的生物学通路中，如内质网相关过程和泛素介导的蛋白水解。选取了几种潜在底物进行验证；事实上，主要蛋白质均发生了棕榈酰化。重要的是，在APT1基因敲除细胞中，它们的棕榈酰化水平发生了明显变化。有趣的是，与野生型细胞相比，APT1基因敲除细胞的增殖显著加快，而APT1过表达可使这一现象得到挽救。
结论：本研究提供了大量APT1的潜在底物，有助于理解其参与的分子功能。
Background: The homeostasis of palmitoylation and depalmitoylation is involved in various cellular processes, the disruption of which induces severe physiological consequences. Acyl-protein thioesterase (APT) and palmitoyl-protein thioesterases (PPT) catalyze the depalmitoylation process. The natural mutation in human PPT1 caused neurodegenerative disease, yet the understanding of APT1 remains to be elucidated. While the deletion of APT1 in mice turned out to be potentially embryonically lethal, the decoding of its function strictly relied on the identification of its substrates.Objective: To determine the potential substrates of APT1 by using the generated human APT1 knockout cell line.Methods : The combined techniques of palmitoyl-protein enrichment and mass-spectrometry were used to analyze the different proteins. Palmitoyl-proteins both in HEK293T and APT1-KO cells were extracted by resin-assisted capture (RAC) and data independent acquisition (DIA) quantitative method of proteomics for data collection.Results: In total, 382 proteins were identified. The gene ontology classification segregated these proteins into diverse biological pathways e.g. endoplasmic reticulum process and ubiquitin-mediated proteolysis. A few potential substrates were selected for verification; indeed, major proteins were palmitoylated. Importantly, their levels of palmitoylation were clearly changed in APT1-KO cells. Interestingly, the proliferation of APT1-KO cells escalated dramatically as compared to that of the WT cells, which could be rescued by APT1 overexpression.Conclusion: Our study provides a large scale of potential substrates of APT1, thus facilitating the understanding of its intervened molecular functions.