PRODUCT MEDIATED REGULATION OF ORNITHINE DECARBOXYLASE

蛋白酶体介导的蛋白质降解是调节细胞功能的核心过程。鸟氨酸脱羧酶（ODC）是多胺生产中的关键酶，参与了一种独特的反馈调节形式，多胺通过诱导蛋白抗酶（AZ）促进其破坏，AZ以ODC为目标，被蛋白酶体降解。具体目的：1)确定ODC和AZ的三维结构，并测试其元素在降解中的作用。拟开展的研究将建立小鼠全长ODC的结构、与ODC结合的AZ部分的复合物、单独的全长AZ以及两种全长蛋白的异源二聚体。将AZ与新鉴定的AZ同源物进行比较，将用于解析降解函数的结构决定因素。x射线模型将用于了解ODC c端结构域的结构，以及该结构域如何以及是否随着AZ的变化而变化。该信息将用于设计ODC末端的重组形式，以确定它们是否与蛋白酶体结合，使用直接关联分析和功能竞争分析。为了区分AZ是单独作为ODC载体，还是作为蛋白酶体的激活剂，将测试AZ的交易功能。2)表征ODC/AZ-蛋白酶体相互作用的阶梯式元件。将鉴定与AZ和ODC相互作用的蛋白酶体成分。与AZ/ODC或相关底物共孵育的蛋白酶体将通过高分辨率电子显微镜检查以定位其位置。ODC和/或AZ与蛋白酶体的化学交联将用于鉴定蛋白质-蛋白质接触。酵母突变体将用于分离26S蛋白酶体的亚组分，并分析其降解ODC的能力。将制作含有阻碍蛋白酶体进入的大块阻塞物的ODC形式，并用于测试降解的方向和进程。3)利用酵母遗传学确定调节ODC降解的细胞成分。我们所测试的多胺诱导的ODC降解的所有特征在动物细胞和酵母中都是相同的，这表明后者将成为研究两者共同特性的信息实验系统。我们已经组装并验证了进行遗传筛选的方法，以分离多胺改变ODC调节的酵母突变体。将鉴定与调节突变体互补的基因，并评估其产物的功能。这些研究将揭示对调节降解重要的细胞成分。

Proteasome-mediated protein degradation is a central process in the regulation of cell function. Ornithine decarboxylase (ODC), a key enzyme in polyamine production, participates in a unique form of feedback regulation, whereby polyamines promote its destruction by inducing the protein antizyme (AZ), which targets ODC for degradation by the proteasome. Specific Aims: 1) Determine the three-dimensional structure of ODC and AZ, and test the function of their elements in degradation. The studies proposed will establish the structure of full length mouse ODC, its complex with the portion of AZ that binds ODC, full length AZ alone and the heterodimer of both full-length proteins. Comparison of AZ to a newly-identified AZ homolog will be used to resolve the structural determinate(s) of degradative function. The x-ray model will be used to understand the structure of the ODC C-terminal domain and how and whether this changes in response to AZ. This information will be exploited to design recombinant forms of the ODC terminus to determine whether these associate with the proteasome, using direct assays of association and functional competition assays. To distinguish whether AZ acts solely as an ODC carrier, or instead or additionally is an activator of the proteasome, AZ will be tested for trans-acting function. 2) Characterize the stepwise elements of ODC/AZ- proteasome interactions. The components of the proteasome that interact with AZ and ODC will be identified. Proteasomes co- incubated with AZ/ODC or related substrates will be examined by high-resolution electron microscopy to localize their position. Chemical crosslinking of ODC and/or AZ to proteasomes will be used to identify protein-protein contacts. Yeast mutants will be used to isolate sub-components of the 26S proteasome and their ability to degrade ODC will be analyzed. Forms of ODC that contain a bulky obstruction that impedes proteasomal entry will be made and used to test the orientation and processivity of degradation. 3) Determine the cellular components that modulate ODC degradation using yeast genetics. All of the features of polyamine-induced ODC degradation that we have tested are identical in animal cells and yeast, suggesting that the latter will be an informative experimental system for studying properties common to both. We have assembled and validated the means to perform genetic screens to isolate yeast mutants altered in their regulation of ODC by polyamines. Genes that complement regulatory mutants will be identified and the function of their products assessed. These studies will reveal cellular components important for regulated degradation.