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作用。它负责 90%的细胞的蛋白质降解需要，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，使免疫系统识别病变细胞。满足这些 生物体的不同需要，蛋白酶体的不同形式存在，并具有相应的特性。有 有限的工具可用于分析细胞中不同类型的蛋白酶体同种型活性。 蛋白酶体介导的蛋白质水解可以通过至少三种不同的途径发生。目前的工具 现有的方法不能有效区分这些蛋白酶体同种型的活性， 被细胞中的其他蛋白酶水解。本项目的目标是设计蛋白酶体异构体选择性 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，以设计我们的 活动探测器在这个项目完成后，我们将有新的蛋白酶体活性探针， 区分泛素依赖性和非依赖性降解的活性。我们还将有一个探测器， 可以检测仅由免疫蛋白酶体介导的蛋白质水解。我们探测器的设计还将包括 重要的二级结构和肽模拟亚基，以防止蛋白酶的非特异性水解， 细胞 区分细胞内蛋白酶体类型的活性将澄清哪种蛋白质降解 这一通路可能成为一种新的治疗靶点。我们预计它们可以用来监测泛素的含量- 在蛋白质积累疾病和血液癌症中的依赖性和非依赖性降解。的 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 探针将提供一种方法来确定免疫蛋白酶体活性对这些疾病的影响程度。我们认为 这些新的探针将填补蛋白酶体领域的空白， 单个蛋白酶体同种型。

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.