RSEARCH PROJECT 2

这项提案的长期目标是确定分子机制和致病性 马洛里小体形成意义。MB的形成涉及许多肝脏疾病的进展，但最值得注意的是酒精性肝病（ALD）和非酒精性脂肪性肝炎（NASH）。这种现象只在人和老鼠身上很明显，所以研究仅限于这两个物种。我们所取得的进展已经允许鉴定MB作为类似于在各种神经退行性疾病如阿尔茨海默氏神经系统缠结中所见的包涵体的侵略性蛋白。我们已经确定MB的组分是细胞角蛋白、天然泛素、突变体泛素（UBB+1）、p62、热休克蛋白70和90、组织转氨酶、微管蛋白和蛋白酶体26 S。此外，我们已经证明，细胞角蛋白进行β折叠转化和过度磷酸化，类似于 侵袭体的形成以及UBB+1（一种由泛素移码突变产生的蛋白质）与MB紧密相关。我们的工作假设是，MB确实是由UBB+1触发的攻击体，并通过抑制细胞角蛋白的蛋白酶体消化引起。目前的建议将确定细胞角蛋白aggresome/MB的形成机制，在体外使用新开发的组织培养模型在我们的实验室。将使用两种模型：1）来自药物致敏小鼠的原代肝细胞培养物，其在培养的第二天形成MB; 2）小鼠肝癌细胞系，其中在用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程和顺序将使用与GFP或RFP融合的UBB+1、C18和p62的荧光探针来确定，所述荧光探针由表达质粒转导。共聚焦荧光显微镜将研究共定位和相互作用的蛋白质在侵略者的动力学。蛋白质转运蛋白Chariot也将用于在体外将大量UBB+1、p62和其他组分引入肝细胞中，以在短得多的持续时间内促进侵袭体形成过程。这将允许实时评估MB发生过程中的事件顺序，并确定初始因素和MB形成过程中涉及的元素。该方法还将用于测试来自酒精喂养小鼠的肝细胞是否以及如何对通过引入不同外源性MB组分触发的MB形成致敏。最后，我们将测试MB形成肝细胞是否易受TNF α（ALD发病机制的核心效应分子）引起的细胞毒性的影响。一旦阐明MB形成的机制和功能意义，就可以设计干预措施来预防其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.