Monitoring and Manipulating the Activity of the Immunoproteasome with Small Molecules

监测和操纵小分子免疫蛋白酶体的活性

• 批准号: 10408807
• 负责人: Darci J Trader
• 金额: $ 37.33万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-02 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10408807
• 关键词: Active Sites; Adaptive Immune System; Affect; Alopecia Areata; Antibodies; Antigens; Antineoplastic Agents; Autoimmune Diseases; Binding; Biological Assay; Bortezomib; Cell Death; Cell membrane; Cell surface; Cells; Clinical Trials; Communication; Complex; Confocal Microscopy; Cytokine Gene; Cytokine Signaling; Data; Disease; Endogenous Factors; Enzymes; Equilibrium; Flow Cytometry; Goals; Health; Hydrolysis; I-antigen; Immune; Immune response; Immune system; Infection; Inflammatory; Insulin-Dependent Diabetes Mellitus; Lead; Lupus; Masks; Methods; Modeling; Molecular Conformation; Monitor; Multienzyme Complexes; Ovalbumin; Peptide Hydrolases; Peptides; Population; Prodrugs; Production; Proteasome Inhibitor; Protein Isoforms; Proteins; Reaction; Reader; Rheumatoid Arthritis; Signal Transduction; Source; T-Cell Activation; T-Lymphocyte; Techniques; Testing; Time; Tissues; Ubiquitin; Virus; Virus Diseases; base; chymotrypsin; combat; cytokine; cytotoxic; design; experimental study; fluorophore; inhibitor; multicatalytic endopeptidase complex; pathogen; peptide I; peptidomimetics; prevent; protein protein interaction; response; screening; small molecule; small molecule inhibitor; stable cell line; tool; treatment response

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.

项目概要 蛋白酶体是一种重要的细胞酶复合物。其主要功能是降解蛋白质 被标记为泛素。当细胞接收到信号（通常是细胞因子）时，就会表达不同的亚型 称为免疫蛋白酶体的蛋白酶体开始产生。免疫蛋白酶体 (iCP) 以与标准蛋白酶体类似的方式降解蛋白质，但其更多产品与 被加载到 MHC-I 复合体中。这些 MHC-I 肽复合物被细胞用来启动适应性 免疫系统通过将肽展示在细胞表面以被免疫细胞识别来做出反应。率 这种类型的免疫系统反应的程度至关重要。例如，当病毒感染细胞时， 重要的是，免疫系统会迅速做出反应，以防止病毒复制过快。然而，如果 当没有感染时，免疫反应就会被触发，T细胞可以开始攻击并破坏健康组织， 导致自身免疫性疾病。 免疫蛋白酶体的抑制最近被探索作为对抗的潜在机制 自身免疫性疾病。假设是，如果 iCP 可以产生较少的 MHC-I 相容性肽， 被激活/发出信号的 T 细胞就越少。然而，当发生病毒感染时，情况恰恰相反，当 MHC-I 相容肽的增加将允许免疫系统快速反应，清除病毒 在它感染更多细胞之前。在本提案中，我们将探讨有多少 iCP 活动会引发何种水平的 MHC- I 在细胞上表达。为了实现这一目标，我们将利用我们最近开发的 iCP 活动探针，该探针可以 使用多种技术用于活细胞和针对特定 MHC-I 抗原复合物的抗体，包括 共焦显微镜和基于读板器的测定。在这些研究正在进行的同时，我们还将利用我们的 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。 完成此处描述的目标后，我们将第一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 活性调节剂，影响自身免疫性疾病和病毒感染。