Monitoring and Manipulating the Activity of the Immunoproteasome with Small Molecules

项目摘要 蛋白酶体是一种重要的细胞酶复合体。它的主要功能是降解具有 被泛素标记了。当细胞接收到信号，通常是细胞因子，表达不同的异构体 的蛋白酶体，称为免疫蛋白酶体，开始产生。免疫蛋白酶体(ICP) 以与标准蛋白酶体相似的方式降解蛋白质，但其更多的产品与 被装载到MHC-I复合体中。这些MHC-I-肽复合体被细胞用来启动适应性 免疫系统通过在细胞表面显示可被免疫细胞识别的多肽来进行免疫反应。利率 而这种免疫系统反应的程度是至关重要的。例如，当病毒感染细胞时，它就是 重要信息：免疫系统反应迅速，防止病毒复制过快。但是，如果 当没有感染时，免疫反应被触发，T细胞可以开始攻击和破坏健康组织， 导致自身免疫性疾病。 最近，免疫蛋白酶体的抑制被探索为一种潜在的对抗 自身免疫性疾病。假设是，如果ICP能够产生较少的MHC-I相容多肽， 被激活/发出信号的T细胞就越少。然而，当病毒感染发生时，情况正好相反，当 增加MHC-I相容多肽将允许快速免疫系统反应，清除病毒 才能感染更多的细胞。在这项提案中，我们将探讨有多少ICP活动会导致MHC- 我在细胞上表达。为了实现这一点，我们将利用我们最近开发的电感耦合等离子体活性探测器，它可以 用于活细胞和特定MHC-I-抗原复合体的抗体，使用各种技术，包括 共聚焦显微镜和基于平板阅读器的分析。虽然这些研究正在进行中，我们也将使用我们的 基于活性的ICP探针，用于筛选可能影响ICP水解的分子，从而导致减少或增加 在MHC-I表达中。 在完成这里描述的目标后，我们将第一次能够量化这种关系 ICP活性与MHC-I表达水平之间的关系。此外，新的小分子抑制物或刺激物 还将发现和研究国际比较方案。长期目标是利用这些新发现的小分子 影响自身免疫性疾病和病毒感染的免疫球蛋白活性调节物。

Project Summary The proteasome is an essential cellular enzyme complex. Its main function is to degrade proteins that have been tagged with ubiquitin. When cells receive a signal, typically a cytokine, the expression of a different isoform of the proteasome, called the immunoproteasome, begins to be produced. The immunoproteasome (iCP) degrades proteins in a similar fashion as the standard proteasome, but more of its products are compatible to be loaded into an MHC-I complex. These MHC-I-peptide complexes are used by cells to initiate the adaptive immune system response by displaying peptides on the cell surface to be recognized by immune cells. The rate and extent of this type of immune system response is critical. For example, when a virus infects cells, it is important the immune system responds rapidly to prevent the virus from replicating too quickly. However, if the immune response is triggered when there is no infection, T-cells can begin to attack and destroy healthy tissue, leading to autoimmune diseases. The inhibition of the immunoproteasome has recently been explored as a potential mechanism to combat autoimmune diseases. The hypothesis is that if less MHC-I compatible peptides can be produced by the iCP, the fewer T-cells will be activated/signaled. However, the opposite is true when a viral infection occurs, when an increase in MHC-I compatible peptides would allow for a rapid immune system response, clearing the virus before it can infect more cells. In this proposal, we will explore how much iCP activity elicits what level of MHC- I expression on a cell. To accomplish this, we will utilize our recently developed iCP-activity probe that can be used in live cells and an antibody to a specific MHC-I-antigen complex using a variety of techniques including confocal microscopy and a plate reader-based assay. While these studies are ongoing, we will also use our activity-based iCP probe to screen for molecules that can affect iCP hydrolysis, leading to a decrease or increase in MHC-I expression. Upon completion of the Aims described here, we will for the first time be able to quantify the relationship between iCP activity and MHC-I expression levels. Additionally, new small molecule inhibitors or stimulators of the iCP will also be discovered and studied. The long-term goal is to use these newly discovered small molecule modulators of iCP activity to affect autoimmune diseases and viral infections.