Recognition of non-ubiquitin signals at the proteasome

描述(申请人提供)：蛋白酶体是在细胞中发现的最大的蛋白质复合体之一。这种依赖于三磷酸腺苷的多亚基蛋白水解酶通过降解精心挑选的靶标来控制许多细胞过程。蛋白酶体功能是所有真核细胞所必需的。蛋白酶体降解途径的缺陷与多种疾病有关，包括癌症、神经退行性疾病、发育障碍、免疫和炎症障碍以及肌肉萎缩。蛋白质是如何被蛋白酶体识别然后被降解的，目前还只有部分了解。如果我们要了解蛋白酶体的作用模式，精确的了解蛋白酶体亚单位的结构，它们之间的相互作用，以及与瞬时辅助因子的关联是至关重要的。这些信息也将作为正在进行的药物设计工作的基础。多泛素化底物靶向蛋白酶体是由几种穿梭蛋白(如Dsk2、RAD23、Ddi1)介导的，它们都含有一个泛素样结构域，与几个蛋白酶体亚基直接相互作用。在这些亚基中，主要的Ubl受体显然是Rpn1。这项拟议研究的目标是了解蛋白酶体如何区分泛素和泛素样信号。我们将通过在生化和结构水平上表征蛋白酶体识别UBL信号所涉及的相互作用的性质和强度来做到这一点。我们将在Rpn1上绘制UBL结合区的图谱，在原子水平上确定专门的UBL受体区域的三维结构，并从结构和生物化学的角度表征这些相互作用的性质和强度。我们将比较UBL的识别，以了解它们之间的差异，以及作为一个群体，它们如何提供与泛素结合指定受体的不同信号。我们将讨论蛋白酶体区分泛素和泛素样蛋白的能力，以及多泛素和泛素在信号传递中的明显冗余。所有的结构预测和配对相互作用将通过生物操作和蛋白质组筛选得到验证。我们将用细胞内相互作用的验证来补充体外表征，测试我们确定的旨在取消或加强成对相互作用的突变体的结果，改变蛋白酶体靶向的层次，并单独观察每个穿梭、UBL信号或受体的广泛蛋白质组学作用。该项目依赖于多种实验方法(生化、生物物理、结构(核磁共振)和蛋白质组学)来揭示蛋白酶体与其底物、靶点和信号的结构-功能关系。这些研究将提供蛋白酶体识别UBL信号所涉及的分子事件的详细图像，触发蛋白酶体的后续事件导致底物降解。

DESCRIPTION (provided by applicant): The proteasome is one of the largest protein complexes to be found in cells. This multi-subunit ATP-dependent protease controls numerous cellular processes by degrading carefully selected targets. Proteasome function is essential for all eukaryotic cells. Defects in the proteasomal degradation pathway are associated with various diseases, including cancers, neurodegenerative disorders, developmental disorders, immune and inflammatory disorders, and muscle wasting. How proteins destined for degradation are recognized by the proteasome and then degraded is only partially understood. Precise knowledge of proteasome subunit architecture, their interactions with each other, and associations with transient auxiliary factors is critical if we are to understand its mode of action. Such information would also serve as the basis for ongoing drug design efforts. Targeting of polyubiquitinated substrates to the proteasome is mediated by several shuttle proteins (e.g., Dsk2, Rad23, Ddi1), all of which contain a ubiquitin-like (UBL) domain that interacts directly with several proteasomal subunits. Of these subunits, the primary UBL-receptor is apparently Rpn1. The goal of the proposed research is to understand how the proteasome differentiates between ubiquitin and ubiquitin-like signals. We will do so by characterizing, at the biochemical and structural levels, the nature and the strength of interactions involved in recognition of the UBL signals by the proteasome. We will map UBL-binding regions on Rpn1, determine the three-dimensional structure of specialized UBL-receptor regions at an atomic level resolution and characterize the nature and the strength of these interactions both structurally and biochemically. We will compare recognition of UBLs to understand how they differ among themselves, and how as a group they pose a distinct signal from ubiquitin which binds to designated receptors. We will address the ability of the proteasome to differentiate between ubiquitin and ubiquitin-like proteins, and the apparent redundancy in signaling by polyubiquitin and UBLs. All structural predictions and pairs-wise interactions will be verified through biological manipulations and proteomic screens. We will complement the in vitro characterization with verification of interactions in cells, test the outcome of mutants designed to abolish or strengthen pair-wise interactions that we identify, alter hierarchy of proteasome targeting, and look at the broad proteomic role of each shuttle, UBL signal, or receptor individually. This project relies on a variety of experimental approaches (biochemical, biophysical, structural (NMR), and proteomic) to reveal the structure-function relationship of the proteasome with its substrates, targets, and signals. These studies will provide a detailed picture of molecular events involved in the recognition of the UBL signals by the proteasome, triggering subsequent events at the proteasome leading to substrate degradation.