Zhang Tao, Meng Jin, Liu Xinli, Zhang Xutao, Peng Xiaojun, Cheng Zhongyi, Zhang Feng
Department of Thoracic Surgery, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, China.
Department of Pharmacology, Key Laboratory of Gastrointestinal Pharmacology of Chinese Materia Medica of the State Administration of Traditional Chinese Medicine, School of Pharmacy, Fourth Military Medical University, Xi'an 710032, China.
Oncotarget. 2017 Oct 31;9(2):1617-1629. doi: 10.18632/oncotarget.22176. eCollection 2018 Jan 5.
ING5 belongs to the Inhibitor of Growth (ING) candidate tumor suppressor family. Previously, we have shown that ING5 inhibits invasiveness of lung cancer cells by downregulating EMT-inducing genes. However, the underlying mechanisms remain unclear. The aim of the study was to use integrated approach involving SILAC labeling and mass spectrometry-based quantitative proteomics to quantify dynamic changes of acetylation regulated by ING5 in lung cancer cells. Here, we have found that ING5 has a profound influence on protein lysine acetylation with 163 acetylation peptides on 122 proteins significantly upregulated and 100 acetylation peptides on 72 proteins downregulated by ING5 overexpression. Bioinfomatic analysis revealed that the acetylated proteins upregulated by ING5 located preferentially in nucleus to cytoplasm and were significantly enriched in transcription cofactor activity, chromatin binding and DNA binding functions; while those downregulated by ING5 located preferentially in cytoplasm rather than nucleus and were functionally enriched in metabolism, suggesting diverse functions of ING5 through differentially regulating protein acetylation. Interestingly, we found ING5 overexpression promotes p300 autoacetylation at K1555, K1558 and K1560 within p300 HAT domain, and two novel sites K1647 and K1794, leading to activation of p300 HAT activity, which was confirmed by accelerated acetylation of p300 target proteins, p53 at k382 and histone H3 at K18. A specific p300 HAT inhibitor C646 impaired ING5-increased acetylation of H3K18 and p53K382, and subsequent expression of p21 and Bax. In conclusion, our results reveal the lysine acetylome regulated by ING5 and provide new insights into mechanisms of ING5 in the regulation of gene expression, metabolism and other cellular functions.
ING5属于生长抑制（ING）候选肿瘤抑制因子家族。此前，我们已经表明ING5通过下调诱导上皮-间质转化（EMT）的基因来抑制肺癌细胞的侵袭性。然而，其潜在机制仍不清楚。本研究的目的是采用基于稳定同位素标记氨基酸细胞培养技术（SILAC）标记和质谱的定量蛋白质组学的综合方法，来量化ING5在肺癌细胞中调节的乙酰化动态变化。在此，我们发现ING5对蛋白质赖氨酸乙酰化有深远影响，ING5过表达使122个蛋白质上的163个乙酰化肽显著上调，72个蛋白质上的100个乙酰化肽下调。生物信息学分析表明，ING5上调的乙酰化蛋白质优先定位于细胞核而非细胞质，并且在转录辅因子活性、染色质结合和DNA结合功能方面显著富集；而ING5下调的那些蛋白质优先定位于细胞质而非细胞核，并且在代谢功能上富集，这表明ING5通过差异调节蛋白质乙酰化具有多种功能。有趣的是，我们发现ING5过表达促进p300 HAT结构域内K1555、K1558和K1560以及两个新位点K1647和K1794处的p300自身乙酰化，导致p300 HAT活性激活，这通过p300靶蛋白p53的K382位点和组蛋白H3的K18位点的乙酰化加速得到证实。一种特异性p300 HAT抑制剂C646损害了ING5增加的H3K18和p53K382乙酰化以及随后p21和Bax的表达。总之,我们的结果揭示了由ING5调节的赖氨酸乙酰化组，并为ING5在基因表达、代谢和其他细胞功能调节中的机制提供了新的见解。
