Development of Activity-Based Chemical Reporters to Differentiate Proteasome Isoforms in Cells

项目摘要 蛋白酶体在真核细胞的正常功能中起着重要的作用。它负责UP 到90%的细胞蛋白质降解需求，控制细胞途径与关键蛋白质的适当平衡 水平，并产生抗原肽，使免疫系统识别患病细胞。为了满足这些要求 生物体的需求不同，不同形式的蛋白酶体存在着相应的特性。确实有 可用于分析细胞中不同类型的蛋白酶体异构体活动的工具有限。 蛋白酶体介导的蛋白质水解至少可以通过三种不同的途径发生。目前的工具 Available不能有效地区分这些蛋白酶体亚型的活性，并且很容易 被细胞中的其他蛋白水解酶降解。本项目的目标是设计具有选择性的蛋白酶体。 活动探测器。我们将广泛利用积累的数据来生产选择性抑制剂来设计我们的 活动探测器。在这个项目完成后，我们将拥有新的蛋白酶体活性探针，可以 区分泛素依赖和非依赖降解的活性。我们还会有一个探测器， 只能检测到由免疫蛋白酶体介导的蛋白质水解。我们的探测器设计还将包括 重要的二级结构和模拟肽的亚基，以防止非特异性的水解酶 细胞。 区分细胞内不同类型的蛋白酶体的活性将有助于阐明哪些蛋白质可降解 通路可作为一种新的治疗方法。我们预计它们可以用来监测泛素的量- 在蛋白质堆积性疾病和血液病中的依赖和独立降解。这个 免疫蛋白酶体最近被认为与多种自身免疫性疾病和I型糖尿病有关。我们的 探针将提供一种方法来确定免疫蛋白酶体活动对这些疾病的影响程度。我们相信 这些新的探针将填补蛋白酶体领域的空白，供人们研究蛋白酶体的活性时使用 个别蛋白酶体亚型。

Project Summary The proteasome plays a significant role in the proper functioning of eukaryotic cells. It is responsible for up to 90% of the cell's protein degradation needs, controls cell pathways with the proper balance of critical protein levels, and produces antigenic peptides to allow the immune system to recognize diseased cells. To meet these diverse needs of the organism, different forms of the proteasome exist with corresponding specialties. There are limited tools available to analyze the different types of proteasome isoform activities in the cells. Proteasome mediated protein hydrolysis can occur through at least three different paths. The tools currently available cannot effectively differentiate between the activity of these proteasome isoforms and are easily hydrolyzed by other proteases in the cell. The goal of this project is to design proteasome isoform-selective activity probes. We will extensively utilize the data accumulated to produce selective inhibitors to design our activity probes. Upon completion of this project, we will have new proteasome activity probes that can differentiate the activity of ubiquitin-dependent and -independent degradation. We will also have a probe that can detect protein hydrolysis mediated by the immunoproteasome only. The design of our probes will also include significant secondary structure and peptidomimetic subunits to prevent non-specific hydrolysis by proteases in cells. Distinguishing the activity of the types of proteasomes within a cell would clarify which protein degradation pathway could be targeted as a new therapy. We anticipate they can be used to monitor the amount of ubiquitin- dependent and -independent degradation in protein accumulation diseases and in hematological cancers. The immunoproteasome has recently been implicated in a variety of autoimmune diseases and type I diabetes. Our probes will provide a way to determine how much immunoproteasome activity affects these diseases. We believe these new probes will fill an empty niche in the proteasome field for when one wants to study the activity of an individual proteasome isoform.