RSEARCH PROJECT 2

这一建议的长期目标是确定分子机制和致病机制 Mallory小体(MB)形成的意义。MBS的形成与多种肝脏疾病的进展有关，但最显著的是酒精性肝病(ALD)和非酒精性脂肪性肝炎(NASH)。这种现象只在人类和老鼠身上才明显，所以研究仅限于这两个物种。我们所取得的进展使我们能够将MB鉴定为一种侵袭性类似于各种神经退行性疾病中所见的包涵体，如阿尔茨海默氏症神经纤维缠绕。我们已经确定MB的组成成分是细胞角蛋白、天然泛素、突变型泛素(UBB 1)、p62、热休克蛋白70和90、组织转谷氨酰胺酶、微管蛋白和蛋白酶体26S。此外，我们还证明了细胞角蛋白经历了β-折叠转化和过度磷酸化，类似于 侵袭体的形成以及UBB 1与MBS密切相关，UBB 1是一种由泛素移码突变产生的蛋白质。我们的工作假设是，MBS确实是由UBB 1触发的侵袭体，由抑制蛋白酶体对细胞角蛋白的消化而引起。目前的建议将利用我们实验室新开发的组织培养模型来确定细胞角蛋白侵袭体/MB的体外形成机制。将使用两种模型：1)药物刺激的小鼠的原代肝细胞培养，在培养的第二天形成MBS；2)小鼠肝癌细胞系，在蛋白酶体抑制剂处理后24小时内形成细胞角蛋白侵袭体。MB组分之间相互作用的时间进程和顺序将使用融合到GFP或RFP的UBB 1、C18和P62荧光探针来确定，这些荧光探针由表达载体转导。共聚焦荧光显微镜将研究侵袭体中蛋白质的共定位和相互作用的动力学。蛋白质转运体Chariot也将在体外将大量的UBB 1、P62和其他成分引入肝细胞，以更短的持续时间促进侵袭体的形成过程。这将有助于实时评估甲基溴形成过程中发生的一系列事件，并确定启动因素和甲基溴形成过程中涉及的要素。这种方法也将被用来测试酒精喂养的小鼠的肝细胞是否以及如何对由引入不同的外源MB成分引发的MB的形成敏感。最后，我们将测试MB形成的肝细胞是否容易受到肿瘤坏死因子α引起的细胞毒性作用，肿瘤坏死因子α是ALD发病机制的核心效应分子。一旦阐明了甲基溴形成的机制和功能意义，就可以设计干预措施来预防它们的形成和可能的相关疾病。

The long-range goal of this proposal is to determine the molecular mechanism and pathogenetic significance of Mallory body (MB) formation. Formation of MBs is implicated in progression of numerous liver diseases but most notably alcoholic liver disease (ALD) and nonalcoholic steatohepatitis (NASH). This phenomenon is evident only in man and mouse so research is limited only to those two species. Progress made by us has allowed identification of the MB as an aggresome analogous to the inclusions seen in various neurologic degenerative diseases such as Alzheimer's neurofibrillary tangles. We have established that the components of the MB are cytokeratins, native ubiquitin, the mutant ubiquitin (UBB+1), p62, heat shock proteins 70 and 90, tissue transglutaminase, tubulin and the proteasome 26S. In addition, we have demonstrated that the cytokeratins undergo beta sheet transformation and hyperphosphorylation, resembling the aggresome formation and that there is a tight association of UBB+1, a protein resulting from a frameshift mutation of ubiquitin, with MBs. Our working hypothesis is that MBs are indeed aggresomes triggered by UBB+1 and caused by inhibition of the proteasomal digestion of cytokeratins. The current proposal will determine the mechanism of cytokeratin aggresome/MB formation in vitro using newly developed tissue culture models in our laboratory. Two models will be used: 1) primary hepatocyte cultures from the drug primed mice that form MBs on day two of culture; 2) mouse hepatoma cell line in which cytokeratin aggresomes form in 24 h after treatment with a proteasome inhibitor. The time course and order of interactions between the MB components will be determined using fluorescent probes of UBB+1, C18 and p62 fused to GFP or RFP that are transduced by expression plasmids. Kinetic of co-localization and interactions of proteins in aggresomes will be studied by confocal fluorescence microscopy. A protein transporter Chariot will also be used to introduce large quantities of UBB+1, p62, and other components into the hepatocytes in vitro to promote the aggresome forming process in much shorter durations. This will allow real-time assessment of the sequence of events in the genesis of MBs and identification of the initialing factor and the elements involved during the progression to MB formation. This method will also be used to test whether and how hepatocytes from alcohol-fed mice are sensitized to MB formation triggered by introduction of different exogenous MB components. Lastly, we will test whether MB forming hepatocytes are vulnerable to cytotoxicity caused by TNFalpha, the effector molecule central to the pathogenesis of ALD. Once the mechanism and functional significance of MB formation are elucidated, interventions can be designed to prevent their formation and possibly associated diseases.