Organelle Dysfunction and Apoptosis in Liver Epithelia

肝上皮细胞器功能障碍和细胞凋亡

• 批准号: 8250599
• 负责人: GREGORY J. GORES
• 金额: $ 34.58万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8250599
• 关键词: Address; Apoptosis; Attenuated; BCL2 Gene Translocation; BIM Bcl-2-binding protein; Binding; Biochemical; Biological; Caspase; Cathepsins; Cell Death; Cell Line; Cells; Cessation of life; Cholestasis; Cytosol; Data; Development; Epithelium; Family; Functional disorder; Hepatic; Hepatocyte; Human; Injury; Ligands; Ligation; Liver; Liver Dysfunction; Liver diseases; Lysosomes; Mediating; Membrane; Mitochondria; Modeling; Molecular; Mus; Organelles; Pathway interactions; Peptide Hydrolases; Poison; Process; Protein Binding; Proteins; Proteolipids; Recruitment Activity; Research; Sorting - Cell Movement; TNFSF10 gene; Technology; Testing; Toxic effect; Tumor Necrosis Factor-alpha; Ubiquitination; base; bile duct; cholangiocyte; cytotoxicity; human BIRC2 protein; human disease; in vivo; innovation; interest; killings; mimetics; novel; novel therapeutics; prevent; primary sclerosing cholangitis; protein activation; protein transport; therapeutic target; trafficking; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): The OVERALL OBJECTIVE of this proposal is to define the cellular mechanisms culminating in lethal injury of cholangiocytes and hepatocytes in models relevant to human cholestatic liver diseases (e.g., primary sclerosing cholangitis). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) contributes to both cholangiocyte and hepatocyte injury in models relevant to these liver diseases. Thus, our long term objectives are to understand the cellular and subcellular mechanisms causing cholangiocyte and hepatocyte injury by this death ligand. Our distinctive preliminary data implicate phosphofurin acidic cluster sorting-2 (PACS-2) protein as a critical regulator of TRAIL-induced cytotoxicity by a lysosomal cell death pathway. Cellular inhibitor of apoptosis-1 (cIAP-1) regulates the lysosomal cell death pathway by controlling PACS-2 protein levels. Cholangiocytes appear to be intrinsically sensitive to TRAIL killing via this pathway in vivo, whereas hepatocytes in vivo only become sensitive to TRAIL toxicity during cholestasis. Based upon this preliminary data, we propose the novel CENTRAL HYPOTHESIS that TRAIL induces liver cell death by a lysosomal pathway dependent upon PACS-2, and cholangiocytes, which express less cIAP-1 than hepatocytes, are preferentially sensitive to this pathway in vivo. We will now employ current and complementary molecular, biochemical and cell biological approaches to ascertain how TRAIL triggers this organelle-based pathway of apoptosis. Our proposal has three SPECIFIC AIMS. FIRST, we will directly test the hypothesis that PACS-2 mediates lysosomal membrane permeabilization during TRAIL cytotoxicity by: a) binding Bim, a BH3-only protein, which recruits and activates Bax, a potent proapoptotic protein of the Bcl-2 family; and b) facilitating Bax oligomerization within lysosomal membranes which causes lysosomal disruption. SECOND, we will directly test the hypothesis that cIAP-1, an E3 ligase, modulates TRAIL cytotoxicity by: a) regulating PACS- 2 protein levels via an ubiquitination-dependent mechanism resulting in its proteasomal degradation; and b) by regulating the lysosomal cell death pathway through control of PACS-2 protein levels. FINALLY, we will directly test the hypothesis that: PACS-2 deletion is salutary in models relevant to cholestatic liver injury by: a) reducing TRAIL-mediated cholangiocyte injury in vivo in a model reminiscent of primary sclerosing cholangitis; and b) by reducing hepatocyte injury in vivo during obstructive cholestasis in the bile duct ligated mouse. The proposal is innovative as it tests new concepts for TRAIL cytotoxicity using sophisticated technologies. The information generated will provide a framework for the development of novel therapeutic strategies effective for attenuating TRAIL-mediated human liver injury during cholestasis. PUBLIC HEALTH RELEVANCE: The application examines the cellular mechanisms by which TRAIL, a natural protein, causes cell death. We propose that TRAIL induces cell death by disrupting a key cellular organelle referred to as the lysosome. Lysosome disruption results in the release of toxic compounds into the cell culminating in cellular demise. The results of these studies are germane to mechanisms of liver injury in a variety of human diseases.

描述(由申请人提供)：本提案的总体目标是在与人类胆汁淤积性肝病(例如，原发性硬化性胆管炎)相关的模型中确定导致胆管细胞和肝细胞致死性损伤的细胞机制。在与这些肝病相关的模型中，肿瘤坏死因子相关的凋亡诱导配体(TRAIL)对胆管细胞和肝细胞的损伤都有贡献。因此，我们的长期目标是了解这种死亡配体导致胆管细胞和肝细胞损伤的细胞和亚细胞机制。我们独特的初步数据表明，磷酸呋喃酸性簇分类-2(PACS-2)蛋白是TRAIL通过溶酶体细胞死亡途径诱导的细胞毒性的关键调节因子。细胞凋亡抑制因子-1(CIAP-1)通过调控PACS-2蛋白水平调控溶酶体细胞死亡途径。体内的肝细胞对TRAIL的毒性只有在胆汁淤积时才变得敏感，而体内的胆管细胞似乎对TRAIL的杀伤具有内在的敏感性。基于这一初步数据，我们提出了一个新的中心假设，即TRAIL通过依赖于PACS-2的溶酶体途径诱导肝细胞死亡，而表达CIAP-1的胆管细胞在体内对这一途径优先敏感。我们现在将使用现有的和互补的分子、生化和细胞生物学方法来确定TRAIL是如何触发这一基于细胞器的凋亡途径的。我们的建议有三个具体目标。首先，我们将直接验证PACS-2在TRAIL细胞毒作用过程中介导溶酶体膜通透性的假设：a)结合Bim，一个仅与BH3结合的蛋白，它招募并激活Bax，一个强有力的促凋亡蛋白；b)促进溶酶体膜内Bax的寡聚，从而导致溶酶体膜的破坏。其次，我们将直接验证CIAP-1，一种E3连接酶，通过以下方式调节TRAIL细胞毒性的假设：a)通过泛素化依赖的机制调节PACS-2蛋白水平，导致其蛋白酶体降解；b)通过控制PACS-2蛋白水平调节溶酶体细胞死亡途径。最后，我们将直接测试这一假设：在与胆汁淤积性肝损伤相关的模型中，Pacs-2缺失是有益的：a)在让人想起原发性硬化性胆管炎的模型中减少TRAIL介导的体内胆管细胞损伤；以及b)在胆管结扎的小鼠梗阻性胆汁淤积期间减少体内肝细胞损伤。该提案是创新的，因为它使用复杂的技术测试新概念的TRAIL细胞毒性。所产生的信息将为开发有效减轻TRAIL介导的人胆汁淤积期肝损伤的新治疗策略提供一个框架。 与公共健康相关：该应用程序检查TRAIL(一种天然蛋白质)导致细胞死亡的细胞机制。我们认为TRAIL通过破坏被称为溶酶体的关键细胞器来诱导细胞死亡。溶酶体的破坏会导致有毒化合物释放到细胞内，最终导致细胞死亡。这些研究的结果与多种人类疾病的肝损伤机制密切相关。