Nakata Shiori, Watanabe Tadashi, Nakagawa Koji, Takeda Hiroshi, Ito Akihiro, Fujimuro Masahiro
Department of Cell Biology, Kyoto Pharmaceutical University, Misasagi-Shichonocho 1, Yamashinaku, Kyoto, 607-8412, Japan.
Department of Pathophysiology and Therapeutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12 Nishi-6, Kitaku, Sapporo, 060-0812, Japan.
Biochem Biophys Res Commun. 2016 Mar 25;472(1):46-52. doi: 10.1016/j.bbrc.2016.02.057. Epub 2016 Feb 17.
Polyubiquitination plays key roles in proteasome-dependent and independent cellular events, whereas monoubiquitination is involved in gene expression, DNA repair, protein-protein interaction, and protein trafficking. We previously developed an FK2 antibody, which specifically recognizes poly-Ub moieties but not free Ub. To elucidate the role of Ub conjugation in response to cellular stress, we used FK2 to investigate whether chemical stress (rapamycin, ethanol, or hydroxyurea), ER stress (thapsigargin or tunicamycin), heat shock or DNA damage (H2O2 or methyl methanesulfonate) affect the formation of Ub conjugates including histone H2A (hH2A) ubiquitination. First, we found that all forms of stress tested increased poly-ubiquitinated proteins in HeLa cells. Furthermore, rapamycin and hydroxyurea treatment, and ER stress increased ubiquitination of hH2A, while methyl methanesulfonate (MMS) treatment induced deubiquitination of hH2A. The ethanol and H2O2 treatments, and heat shock transiently induced hH2A de-ubiquitination, although deubiquitinated hH2A were ubiquitinated again by subsequent cultivation. We also revealed that FK2 reacts with not only polyubiquitinated proteins but also mono-ubiquitinated hH2A. With the exception of MMS, all forms of stress tested increased the acetylation of K5-hH2A, K9-hH3 and K8-hH4 in addition to ubiquitination. K118 and K119 of hH2A were ubiquitinated in cells under normal conditions, and K119 was the major ubiquitination site. The MMS-treatment and heat shock induced the deubiquitination of both K118 and K119-histone H2A. Interestingly, MMS treatment did not affect cell HeLa cell viability expressing double-mutant hH2A (KK118,119AA-hH2A), while heat shock slightly but significantly decreased viability of double-mutant hH2A expressing cells, indicating that ubiquitination of both sites associates with recovery from heat shock but not MMS treatment. Thus, we characterized FK2 reactivity and demonstrated that various stresses alter the ubiquitination status, particularly ubiquitinated hH2A and with histone acetylation, and highlight the physiological importance of hH2A ubiquitination after exposure to stress stimuli.
多聚泛素化在蛋白酶体依赖性和非依赖性细胞事件中发挥关键作用，而单泛素化则参与基因表达、DNA修复、蛋白质-蛋白质相互作用和蛋白质运输。我们之前开发了一种FK2抗体，它能特异性识别多聚泛素部分，但不能识别游离泛素。为了阐明泛素缀合在细胞应激反应中的作用，我们使用FK2来研究化学应激（雷帕霉素、乙醇或羟基脲）、内质网应激（毒胡萝卜素或衣霉素）、热休克或DNA损伤（过氧化氢或甲磺酸甲酯）是否会影响包括组蛋白H2A（hH2A）泛素化在内的泛素缀合物的形成。首先，我们发现所有测试的应激形式都增加了HeLa细胞中多聚泛素化蛋白的含量。此外，雷帕霉素和羟基脲处理以及内质网应激增加了hH2A的泛素化，而甲磺酸甲酯（MMS）处理诱导了hH2A的去泛素化。乙醇和过氧化氢处理以及热休克短暂诱导了hH2A的去泛素化，尽管去泛素化的hH2A在随后的培养中再次被泛素化。我们还发现FK2不仅与多聚泛素化蛋白反应，还与单泛素化的hH2A反应。除了MMS，所有测试的应激形式除了增加泛素化外，还增加了K5-hH2A、K9-hH3和K8-hH4的乙酰化。在正常条件下，hH2A的K118和K119在细胞中被泛素化，且K119是主要的泛素化位点。MMS处理和热休克诱导了K118和K119组蛋白H2A的去泛素化。有趣的是，MMS处理不影响表达双突变hH2A（KK118,119AA-hH2A）的HeLa细胞活力，而热休克略微但显著降低了表达双突变hH2A细胞的活力，这表明两个位点的泛素化与从热休克中恢复有关，但与MMS处理无关。因此，我们对FK2的反应性进行了表征，并证明各种应激会改变泛素化状态，特别是泛素化的hH2A以及组蛋白乙酰化，并突出了应激刺激后hH2A泛素化的生理重要性。
