Tachibana Shingo, Otaki Yoichiro, Watanabe Tetsu, Goto Jun, Ochi Haruki, Tanaka Toshiaki, Ono Hiroe, Yamaguchi Ryuhei, Sato Junya, Takahashi Hiroki, Arimoto Takanori, Goto Kaoru, Watanabe Masafumi
Department of Cardiology, Pulmonology, and Nephrology Yamagata University School of Medicine Yamagata Japan.
Institute for Promotion of Medical Science Research, Faculty of Medicine Yamagata University Yamagata Japan.
J Am Heart Assoc. 2025 Jan 7;14(1):e035608. doi: 10.1161/JAHA.124.035608. Epub 2024 Dec 24.
Doxorubicin-induced cardiotoxicity is still an important medical problem associated with a high mortality rate in cancer survivors. p53 plays a key role in doxorubicin-induced cardiotoxicity. Diacylglycerol kinase ζ (Dgkζ), a 130-kDa enzyme abundant in cardiomyocytes, regulates the p53 protein expression level in neurons. To elucidate the mechanism of doxorubicin-induced cardiotoxicity, we focused on the functional role of Dgkζ and its interaction with heat shock protein 70 (Hsp70)-related ubiquitin E3 ligases such as E6-associated protein (E6ap) and C-terminus of Hsp70-interacting protein.
Protein interactions of Dgkζ with Hsp70 and E6ap were confirmed by immunoprecipitation, but not C-terminus of Hsp70-interacting protein. We administered doxorubicin in cardiac-specific overexpression of Dgkζ transgenic (Dgkζ-Tg) mice and wild-type littermates. Dgkζ-Tg mice showed lower p53 protein expression levels, preserved cardiac function, and improved survival rates compared with wild-type littermates after doxorubicin administration. RNA sequence analysis of myocardial tissues from Dgkζ-Tg after doxorubicin stimulation identified encoding Hsp70 as the differentially expressed gene. Dgkζ overexpression increased proteasomal p53 degradation and attenuated cardiomyocyte apoptosis after doxorubicin stimulation in cardiomyocytes, which was reversed by knockdown of E6ap. Dgkζ interacted with E6ap through ankyrin-like repeats. The overexpression of mutant Dgkζ, lacking ankyrin-like repeats, failed to inhibit p53 protein expression after doxorubicin stimulation. In Dgkζ-overexpressing cardiomyocytes, expression levels of p53 and caspase-3 were increased by knockdown of the C-terminus of Hsp70-interacting protein.
We demonstrated for the first time that Dgkζ augments p53 ubiquitin-proteasome degradation and ameliorates doxorubicin-induced cardiotoxicity by interacting with Hsp70 and E3 ligases such as E6ap and C-terminus of Hsp70-interacting protein.
阿霉素诱导的心脏毒性仍是一个重要的医学问题，与癌症幸存者的高死亡率相关。p53在阿霉素诱导的心脏毒性中起关键作用。二酰基甘油激酶ζ（Dgkζ）是一种在心肌细胞中丰富的130 kDa酶，可调节神经元中p53蛋白的表达水平。为了阐明阿霉素诱导心脏毒性的机制，我们聚焦于Dgkζ的功能作用及其与热休克蛋白70（Hsp70）相关的泛素E3连接酶（如E6相关蛋白（E6ap）和Hsp70相互作用蛋白的C末端）的相互作用。
通过免疫沉淀证实了Dgkζ与Hsp70和E6ap之间的蛋白质相互作用，但未证实与Hsp70相互作用蛋白的C末端的相互作用。我们对心脏特异性过表达Dgkζ的转基因（Dgkζ-Tg）小鼠和野生型同窝小鼠给予阿霉素。与野生型同窝小鼠相比，Dgkζ-Tg小鼠在给予阿霉素后显示出较低的p53蛋白表达水平、保留的心脏功能和提高的存活率。对阿霉素刺激后的Dgkζ-Tg心肌组织进行RNA序列分析，确定编码Hsp70为差异表达基因。在心肌细胞中，Dgkζ过表达增加了蛋白酶体对p53的降解，并减轻了阿霉素刺激后的心肌细胞凋亡，而E6ap的敲低可逆转这种情况。Dgkζ通过锚蛋白样重复序列与E6ap相互作用。缺乏锚蛋白样重复序列的突变型Dgkζ过表达在阿霉素刺激后未能抑制p53蛋白表达。在过表达Dgkζ的心肌细胞中，敲低Hsp70相互作用蛋白的C末端可增加p53和caspase-3的表达水平。
我们首次证明，Dgkζ通过与Hsp70以及E6ap和Hsp70相互作用蛋白的C末端等E3连接酶相互作用，增强p53泛素-蛋白酶体降解并改善阿霉素诱导的心脏毒性。
