INVOLVEMENT OF UBIQUITINATED PROTEINS IN 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降解途径 蛋白水解独立发挥作用。 邓恩博士和其他人最近 揭示了应激细胞中这些降解途径之间的联系。 除了更高分子量的泛素结合物，Dunn博士 在自噬细胞中发现了两种主要的泛素化蛋白（Ub 60和Ub 68）， 营养胁迫细胞的空泡（AV）。 然而，有缺陷的ts 20细胞 在蛋白质泛素化中，不能降解蛋白质 应激导致缺乏泛素化的AV的积累 proteins. 待检验的假设是：蛋白质泛素化 途径是自噬介导的蛋白质的重要组成部分 降解 为了验证这一假设， 底物蛋白和/或包含自噬机制的蛋白 对溶酶体蛋白水解必不可少。 受体 Ub 68的蛋白，ap 40，将被克隆并在其上标记表位（c-myc）。 C-末端，并且赖氨酸将通过 定点诱变。 这些重组蛋白将被稳定地 转染到肝癌细胞中，并在 非应激和营养应激条件下。 生化和 体内和体外形态学分析将评估 降解这些蛋白质，并研究泛素化的作用， ap 40在AV内的隔离和降解。 周转率 将在两个正常细胞系中比较ap 40-myc和ap 40 lys-Arg-myc的表达， 两个泛素化突变细胞系和一个回复突变细胞系， 用重组蛋白质转染。 最后，影响 从突变体细胞分离的AV的泛素化对它们的能力， 将评估螯合和降解AP 40。 这些研究将进一步 描述泛素介导的蛋白质 降解和应激诱导的自噬。

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.