Autophagic dysfunction in the liver enhances the expression of insoluble nuclear proteins 14-3-3ζ and importin α4
Autophagic dysfunction in the liver enhances the expression of insoluble nuclear proteins 14-3-3ζ and importin α4
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肝脏自噬功能障碍增强不溶性核蛋白 14-3-3ζ 和输入蛋白 α4 的表达
DOI：
10.1016/j.lfs.2022.120491
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Kenichi Ikejima
中科院分区：
文献类型：
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作者：
Kousuke Izumi ;Shunhei Yamashina ;Tsutomu Fujimura;Sumio Watanabe ;Kenichi Ikejima
自噬功能障碍与包括非酒精性脂肪性肝病（NAFLD）在内的多种肝病的进展相关。然而，评价自噬功能的血清标志物还未见报道。高度不溶性核蛋白参与许多细胞功能，是癌症的潜在诊断标志物。我们进行了一个蛋白质组学分析的肝细胞核不溶性部分，以确定新的自噬相关的诊断biologicals.Main方法的不溶性核蛋白组分提取的肝Atg 7 F/F，Atg 7 F/F：白蛋白Cre（肝细胞特异性自噬缺陷小鼠），C57 BL/6 J，和KKAy（NAFLD模型）小鼠。通过双向电泳分离蛋白质，并通过银染色可视化。使用质谱法鉴定蛋白质点。免疫荧光共聚焦显微镜检测肝细胞中蛋白质的定位。关键发现不溶性核蛋白14-3-3 β和importin α4的水平在肝自噬功能障碍后上调，并且在血清中可检测到。在正常情况下，这些蛋白质主要分布在细胞质中，而自噬功能障碍诱导它们移位到细胞核。与自噬抑制剂一起孵育上调了这些蛋白在原代肝细胞不溶性核部分中的表达。用EGF或胰岛素处理增强了核不溶性部分中14-3-3 β的表达;相反，加入雷帕霉素下调了14-3-3 β的表达。结果表明，在细胞核不溶性部分，经衣霉素或过氧化氢孵育后，Importin α4表达增加，14-3-3 β和Importin α4作为核不溶性蛋白的积累可能与自噬功能障碍有关。我们的研究结果表明，这些蛋白质可能是有用的诊断生物标志物的肝脏疾病的自噬性疾病。
AimsAutophagic dysfunction is associated with the progression of various liver diseases, including nonalcoholic fatty liver disease (NAFLD). However, serum markers for evaluating autophagic function have not been reported. Highly insoluble nuclear proteins participate in many cellular functions and are potential diagnostic markers for cancer. We performed a proteomic analysis of the hepatic nuclear insoluble fraction to identify novel autophagy-related diagnostic biomarkers.Main methodsThe insoluble nuclear protein fraction was extracted from the livers ofAtg7F/F,Atg7F/F:alb-Cre (hepatocyte-specific autophagy-deficient mice), C57BL/6 J, and KKAy(NAFLD model) mice. Proteins were separated by two-dimensional electrophoresis and visualized by silver staining. Protein spots were identified using mass spectrometry. The localization of proteins in hepatocytes was verified by immunofluorescence using a confocal microscope.Key findingsThe levels of insoluble nuclear proteins 14-3-3ζ and importin α4 were upregulated following hepatic autophagy dysfunction and were detectable in serum. Under normal conditions, these proteins are mainly distributed in the cytoplasm, whereas autophagic dysfunction induces their translocation to the nucleus. Incubation with an autophagy inhibitor up-regulated these proteins expression in the insoluble nuclear fraction of primary hepatocytes. Treatment with EGF or insulin enhanced 14-3-3ζ expression in the nuclear insoluble fraction; in contrast, the addition of rapamycin downregulated 14-3-3ζ expression. Importin α4 expression was increased in the nuclear insoluble fraction after incubation with tunicamycin or hydrogen peroxide.SignificanceAccumulation of 14-3-3ζ and importin α4 as nuclear-insoluble proteins may be associated with autophagic dysfunction. Our findings indicate that these proteins might be useful diagnostic biomarkers for liver diseases with autophagic disorders.