Toward understanding the molecular mechanisms that regulate intermediate filament keratin functions in hepatocytes

中间丝具有微管和肌动蛋白微丝，它们是大多数哺乳动物细胞的主要细胞骨架成分。在许多人类慢性肝病如酒精性肝炎、肝豆状核变性和肥胖症中，K8/18IF的改变和含有角蛋白的聚集体的形成称为Mallory-Denk小体是常见的。最近的研究表明，K8/18在保护肝细胞免受慢性应激性肝损伤中起重要作用。 我们项目的长期目标是确定K8/18在肝细胞中发挥保护作用的分子机制。更具体地说，我们想要测试的假设是，K8/18在维持促和抗凋亡信号分子之间的微妙平衡方面起着关键的调节作用。这项建议的短期目标是：1)进行一项功能研究，直接评估角蛋白在抗细胞凋亡途径PI3K/Akt中的作用。我们将确定在细胞对Fas和肿瘤坏死因子诱导的细胞凋亡的反应中，K8/18的动态调节(表达，特定位点的磷酸化)如何影响Akt通路。2)越来越多的证据表明，角蛋白的位点和模式特异性磷酸化在保护肝细胞免受应激中起着关键作用。因此，我们想要用蛋白质组学的方法来描述K8/18在对照肝细胞和用肝毒药物灰黄霉素处理的小鼠肝细胞中的磷酸化模式。这项研究之后将对特定的K8/18磷酸化组合进行功能分析。 角蛋白参与的信号通路的特征以及在肝细胞对凋亡信号的反应中影响角蛋白的修饰(翻译后修饰，与其他蛋白质的结合)的研究将是理解角蛋白完成其功能的分子机制的重要一步。这将直接引导我们进一步研究角蛋白与细胞信号之间的相互作用，以及角蛋白如何影响疾病的发病机制。

Intermediate filaments (IFs) are with microtubules and actin microfilaments the major cytoskeletal components of most mammalian cells. Modifications in K8/18 IFs organization and formation of keratin containing aggregates called Mallory-Denk bodies are common to many human chronic liver diseases such as alcoholic hepatitis, Wilson's disease and liver steatosis in obesity. Recent studies have shown evidences that K8/18 play an important role in the protection of hepatocytes against chronic stress-induced liver injuries. The long-term objective of our project is to determine the molecular mechanism by which K8/18 accomplish their protective role in hepatocytes. More specifically we want to test the hypothesis that K8/18 act as a key regulator in maintaining the delicate balance between pro and anti-apoptotic signals molecules. The short term objective of this proposal is to 1) perform a functional study to directly asses the role of keratins in the anti-apoptotic pathway PI3K/Akt. We will determine how modulation of K8/18 dynamic (expression, phosphorylation on specific sites) affects Akt pathway during the response of cell to Fas and TNF induced apoptosis. 2) There is accumulating evidence that sites and pattern- specific phosphorylation of keratin play a key role in the protection of hepatocytes against stress. Thus, we want to characterize by a proteomic approach the phosphorylation patterns of K8/18 in control hepatocytes and in hepatocytes from mice treated with the hepatotoxic drug griseofulvin. This study will be followed by a functional analysis of the specific K8/18 phosphorylation combination. The characterization of the signaling pathway in which keratins are involved and the study of the modifications that affect keratins (post-translational modification, association with other proteins) during the response of hepatocytes to apoptotic signal will constitute an important step in our understanding of the molecular mechanisms by which keratins accomplish their functions. This will directly lead us to further research on the cross-talk between keratins and cell signaling, and on how keratins affect disease pathogenesis.