Defining how the Proteasome Recognizes its Ubiquitylated Substrates

描述（由申请人提供）：泛素蛋白酶体途径调节一系列惊人的细胞事件，并且在细胞的整个生命周期中仍然是必不可少的；它的功能障碍与癌症和神经退行性疾病等威胁性疾病有关。该途径分为两个阶段：底物泛素化，最终导致多泛素与蛋白质底物的共价附着；蛋白酶体降解，导致底物降解为免疫活性肽。这两个事件之间的联系需要泛素受体。该项目的目标是确定连接底物泛素化和蛋白酶体降解的机制途径，因此将重点放在泛素受体上。我们使用核磁共振来确定与多泛素络合的蛋白酶体和非蛋白酶体受体的结构。这些研究与荧光光谱和分析超离心相辅相成，以建立结合亲和力和化学计量学。最终，我们的结构数据的后果是探索功能分析。由于新的泛素受体和蛋白酶体成分仍在揭示，对蛋白酶体如何捕获和处理其底物的机制理解尚处于起步阶段。事实上，我们所在的研究团队已经确定了一种新的蛋白酶体泛素受体，作为该提案的一部分，我们确定了它与多泛素复合物的结构以及它与其他蛋白酶体组分相互作用的功能含义。通过核磁共振，我们可以很容易地监测多泛素和多个结合伙伴之间的动态相互作用，这是一个很大的资产，因为泛素受体相互结合，这种相互作用可能为底物穿梭到蛋白酶体和蛋白酶体内部提供有效的机制。我们确定了各种受体如何调节彼此与泛素的相互作用，并影响其他蛋白酶体事件，特别是去泛素化。最终，我们的结果将提供关于蛋白酶体如何捕获其泛素化底物的基本信息。公共卫生相关性：作为基因组完整性重要过程的关键调节因子，如细胞周期调节和基因表达，泛素蛋白酶体途径在治疗癌症和神经退行性疾病方面具有许多治疗可能性。值得注意的是，硼替佐米/Velcade抑制蛋白酶体是多发性骨髓瘤患者唯一可用的治疗方法；然而，蛋白酶体抑制的副作用是严重的。我们的研究有望提供关于泛素化底物如何穿梭到蛋白酶体和蛋白酶体内部的基本信息。这些知识是合理设计特定蛋白质底物抑制剂的第一步，从长远来看，这些抑制剂可以在临床上使用，副作用很少。

DESCRIPTION (provided by applicant): The ubiquitin proteasome pathway regulates an astounding array of cellular events and remains essential throughout the life cycle of a cell; its dysfunction is associated with ailments as threatening as cancer and neurodegenerative diseases. The pathway functions in two stages, substrate ubiquitination, which culminates in covalent attachment of polyubiquitin to protein substrates, and proteasomal degradation, which results in the degradation of substrate into immunocompetent peptides. The connection between these two events requires ubiquitin receptors. The goal of this project is to determine the mechanistic pathways connecting substrate ubiquitination to proteasomal degradation and the aims therefore focus on ubiquitin receptors. We use NMR to determine the structure of proteasomal and non-proteasomal receptors complexed with polyubiquitin. These studies are complemented with fluorescence spectroscopy and analytical ultracentrifugation to establish binding affinity and stoichiometry. Ultimately, the consequences of our structural data are explored by functional assays. A mechanistic understanding of how the proteasome captures and processes its substrates is in its infancy, as new ubiquitin receptors and proteasome components are still being revealed. In fact, we are part of a research team that has identified a new proteasomal ubiquitin receptor, and as part of this proposal, we determine its structure complexed with polyubiquitin as well as the functional implications of its interaction with other proteasome components. By using NMR, we can readily monitor dynamic interactions between polyubiquitin and multiple binding partners, which is a large asset, as ubiquitin receptors bind each other and such interactions are likely to provide an effective mechanism for shuttling substrates to and within the proteasome. We determine how the various receptors modulate each other's interactions with ubiquitin and affect other proteasomal events especially deubiquitination. Ultimately, our results will provide fundamental information on how the proteasome captures its ubiquitinated substrates. Public Health Relevance: As a key regulator of processes important for genome integrity, such as cell cycle regulation and gene expression, the ubiquitin proteasome pathway harbors numerous therapeutic possibilities for treating cancer and neurodegenerative diseases. Notably, inhibition of the proteasome by bortezomib/Velcade is the only treatment available to patients with multiple myeloma; however, the side effects of proteasome inhibition are severe. Our research is expected to provide fundamental information on how ubiquitinated substrates are shuttled to and within the proteasome. Such knowledge is the first step towards rationally designing inhibitors for specific protein substrates, which in the long-term could be used clinically with few side effects.