INVOLVEMENT OF UBIQUITINATED PROTEINS IN AUTOPHAGY

泛素化蛋白参与自噬

• 批准号: 2139024
• 负责人: WILLIAM A DUNN
• 金额: $ 13.99万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-01-01 至 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2139024
• 关键词: HTC cell; aldehyde lyase; autophagy; human genetic material tag; lysosomes; molecular biology; molecular cloning; mutant; proteolysis; recombinant proteins; site directed mutagenesis; stress; transfection; ubiquitin

DESCRIPTION (Adapted from the applicant's abstract): Until recently it was thought that the ubiquitin and lysosomal protein degradation pathways of proteolysis functioned independently. Dr. Dunn and others have recently uncovered a linkage between these degradation pathways in stressed cells. In addition to higher molecular weight ubiquitin conjugates, Dr. Dunn has found two major ubiquitinated proteins (Ub60 and Ub68) within autophagic vacuoles (AVs) of nutrient-stressed cells. However, ts20 cells, defective in protein ubiquitination, are unable to degrade proteins in response to stress resulting in the accumulation of AVs that lack ubiquitinated proteins. The hypothesis to be tested is: The protein ubiquitination pathway is an essential component of autophagy-mediated protein degradation. To test this hypothesis whether or not ubiquitination of substrate proteins and/or proteins that comprise the machinery of autophagy are essential for lysosomal proteolysis will be examined. The acceptor protein of Ub68, ap40, will be cloned and epitope-tagged (c-myc) at its C-terminus, and lysines will be converted to arginines through site-directed mutagenesis. These recombinant proteins will be stably transfected into hepatoma cells and their turnover measured under nonstresses and nutrient-stressed conditions. Biochemical and morphological analyses in vivo and in vitro will evaluate the site of degradation of these proteins and investigate the role of ubiquitination of ap40 in its sequestration and degradation within AVs. The turnover rates of ap40-myc and ap40 lys-arg-myc will be compared in two normal cell lines, two ubiquitination mutant cell lines, and a revertant cell line that have been transfected with the recombinant proteins. Finally, the effects of ubiquitination of AVs isolated from the mutant cells on their ability to sequester and degrade ap40 will be evaluated. These studies will further characterize the relationship between ubiquitin-mediated protein degradation and stress-induced autophagy.

描述(改编自申请人的摘要)：直到最近 认为泛素和溶酶体蛋白的降解途径 蛋白水解酶独立发挥作用。邓恩博士和其他人最近 在应激细胞中发现了这些降解途径之间的联系。 除了更高分子量的泛素结合物，邓恩博士还 在自噬中发现了两种主要的泛素化蛋白(Ub60和Ub68) 营养胁迫细胞的空泡(AVs)。然而，TS20电池，有缺陷 在蛋白质泛素化中，不能降解蛋白质以响应 应激导致缺乏泛素化的AVs的积累 蛋白质。需要检验的假设是：蛋白质泛素化 途径是自噬介导的蛋白质的重要组成部分 退化。为了检验这一假说是否泛素化 底物蛋白质和/或构成自噬机制的蛋白质 对于溶酶体的蛋白分解是必不可少的，将被检测。接受者 Ub68蛋白ap40将被克隆并在其上标记(c-myc) C末端，赖氨酸将通过以下途径转化为精氨酸 定点突变。这些重组蛋白将稳定地 转染人肝癌细胞及其周转率的测定 非胁迫和营养胁迫条件。生化和 体内和体外的形态分析将评估 这些蛋白的降解，并研究泛素化在这些蛋白中的作用 AP40在AVs内的封存和降解。流动率 将在两个正常细胞系中比较ap40-myc和ap40lys-arg-myc的差异， 两个泛素化突变细胞系和一个具有 用重组蛋白进行了基因表达。最后，它的影响 从突变细胞中分离的AVs泛素化对其能力的影响 将对隔离和降解ap40进行评估。这些研究将进一步 表征泛素介导的蛋白质之间的关系 降解和应激诱导的自噬。