Monitoring and Manipulating the Activity of the Immunoproteasome with Small Molecules

项目摘要 蛋白酶体是一种必需的细胞酶复合物。它的主要功能是降解蛋白质， 被标记了泛素当细胞接收到信号时，通常是细胞因子，不同亚型的表达 免疫蛋白酶体开始产生。免疫蛋白酶体（iCP） 以类似于标准蛋白酶体的方式降解蛋白质，但它的更多产物与 被装载到MHC-I复合体中。这些MHC-I-肽复合物被细胞用来启动适应性免疫应答。 通过在细胞表面上展示肽以被免疫细胞识别来产生免疫系统应答。率 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， 重要的是，免疫系统迅速作出反应，以防止病毒复制过快。但如果 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 免疫蛋白酶体的抑制最近已被探索为一种潜在的机制，以打击 自身免疫性疾病假设是如果iCP可以产生较少的MHC-I相容肽， 激活/发信号的T细胞越少。然而，当病毒感染发生时，情况正好相反， MHC-I相容肽的增加将允许快速的免疫系统应答，清除病毒， 才能感染更多的细胞在这个建议中，我们将探讨有多少iCP活动elevated什么水平的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iCP活性探针， 用于活细胞和特异性MHC-I-抗原复合物的抗体 共聚焦显微镜和基于平板读数器的测定。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，用于筛选可影响iCP水解的分子，从而导致iCP水解减少或增加 MHC-I表达。 在完成这里描述的目标后，我们将首次能够量化这种关系 iCP活性和MHC-I表达水平之间的关系。此外，新的小分子抑制剂或刺激剂， iCP也将被发现和研究。长期目标是利用这些新发现的小分子 iCP活性的调节剂，以影响自身免疫性疾病和病毒感染。

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.