RSEARCH PROJECT 2

研究项目2

• 批准号: 6884961
• 负责人: SAMUEL William FRENCH
• 金额: $ 18.16万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6884961
• 关键词: alcoholic liver cirrhosis; conformation; drug metabolism; ethanol; gene expression; heat shock proteins; inclusion body; infrared spectrometry; intermediate filaments; keratin; laboratory mouse; liver cells; liver disorder; northern blottings; posttranslational modifications; protein binding; protein folding; protein glutamine gamma glutamyltransferase; protein metabolism; proteolysis; stress proteins; toxicology; tubulin; ubiquitin; western blottings

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.

该提案的远程目标是确定分子机制和致病性 Mallory体（MB）形成的意义。 MB的形成与众多肝病的进展有关，但最著名的是酒精性肝病（ALD）和非酒精性脂肪性肝炎（NASH）。这种现象只有在人和老鼠中才能明显，因此研究仅限于这两个物种。美国取得的进展允许将MB鉴定为类似于在各种神经系统退行性疾病（例如阿尔茨海默氏症的神经纤维缠结）中的包含物。我们已经确定MB的成分是细胞角蛋白，天然泛素，突变泛素（UBB+1），p62，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，我们已经证明了细胞角蛋白经历β片转化和高磷酸化，类似于 聚集体的形成，并且ubb+1的缔合是由泛素的移料突变与MBS与MBS相关的蛋白质。我们的工作假设是，MBS确实是由UBB+1触发的摄影，并由抑制细胞角蛋白的蛋白酶体消化引起。当前的建议将在我们的实验室中使用新开发的组织培养模型在体外确定细胞角蛋白的细胞角蛋白/MB形成的机制。将使用两种模型：1）在培养第二天形成MB的药物的原发性肝细胞培养物； 2）小鼠肝瘤细胞系在用蛋白酶体抑制剂处理后24小时内形成细胞角蛋白脂肪组。 MB组件之间的时间过程和相互作用的顺序将使用UBB+1，C18和P62的荧光探针融合到GFP或RFP的荧光探针，而GFP或RFP被表达质粒转导。共同定位的动力学和蛋白质中蛋白质的相互作用将通过共聚焦荧光显微镜研究。蛋白质转运蛋白的战车还将用于在体外引入大量的UBB+1，P62和其他成分，并在体外引入肝细胞，以在更短的持续时间内促进整体的形成过程。这将允许对MBS起源中事件的序列进行实时评估，并鉴定出在MB形成过程中涉及的初始因素以及所涉及的元素。该方法还将用于测试是否以及如何通过引入不同的外源MB成分引起的酒精喂养小鼠对MB形成的敏感性。最后，我们将测试形成肝细胞的MB是否容易受到TNFALPHA引起的细胞毒性的影响，Tnfalpha是ALD发病机理的中心效应分子。一旦阐明了MB形成的机制和功能意义，就可以设计干预措施以防止其形成和可能相关的疾病。