PRODUCT MEDIATED REGULATION OF ORNITHINE DECARBOXYLASE

蛋白酶体介导的蛋白质降解是细胞功能调节的中心过程。鸟氨酸脱羧酶(ODC)是多胺产生的关键酶，参与了一种独特的反馈调节，多胺通过诱导蛋白质抗酶(AZ)来促进其破坏，蛋白质抗酶(AZ)靶向ODC被蛋白酶体降解。具体目标：1)确定ODC和AZ的三维结构，并测试其元素在降解过程中的作用。所提出的研究将建立全长小鼠ODC的结构，其与结合ODC的AZ部分、仅全长AZ以及两种全长蛋白的异源二聚体的复合体。AZ与新发现的AZ同系物的比较将被用来解析退化函数的结构定式(S)。X射线模型将被用来了解ODC C-末端结构域的结构，以及这种结构如何以及是否随着AZ的变化而变化。这些信息将被用来设计ODC末端的重组形式，以确定这些重组形式是否与蛋白酶体相关，使用直接结合分析和功能竞争分析。为了区分AZ是仅仅作为ODC携带者，还是作为蛋白酶体的激活剂，AZ将进行反式作用功能测试。2)研究ODC/AZ-蛋白酶体相互作用的阶梯式元件。与AZ和ODC相互作用的蛋白酶体的成分将被确定。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进行检查以定位它们的位置。ODC和/或AZ与蛋白酶体的化学交联将被用来识别蛋白质-蛋白质接触。酵母突变株将用于分离26S蛋白酶体的亚组分，并将分析它们降解ODC的能力。含有阻碍蛋白酶体进入的巨大障碍的ODC形式将被制作并用于测试降解的方向和处理能力。3)利用酵母遗传学确定调节ODC降解的细胞成分。我们测试的多胺诱导的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性质的一个信息丰富的实验系统。我们已经组装并验证了进行遗传筛选以分离酵母突变株的方法，这些突变株在多胺对ODC的调节中发生了变化。将识别与调节突变互补的基因，并评估其产物的功能。这些研究将揭示对调节降解很重要的细胞成分。

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.