Gluten peptide presentation in celiac disease: investigating the role of transglutaminase 2 using novel chemical probes

乳糜泻中的麸质肽呈递：使用新型化学探针研究转谷氨酰胺酶 2 的作用

• 批准号: 10671485
• 负责人: Harrison Anthony Besser
• 金额: $ 4.05万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2024-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10671485
• 关键词: Abdomen; Active Sites; Affect; Amino Acids; Antigen Presentation; Antigen-Presenting Cells; Antigens; Area; Autoimmune Diseases; Binding; Biological; Biological Assay; CD4 Positive T Lymphocytes; Celiac Disease; Cells; Cereals; Characteristics; Chemicals; Clinical; Coculture Techniques; Complex; Confocal Microscopy; Dendritic Cells; Diffusion; Disease; Dose; Endocytosis; Endosomes; Enzyme Inhibitor Drugs; Enzymes; Epidemiology; Event; Extracellular Matrix; Flow Cytometry; Fluorescent Dyes; Fluorescent Probes; Gastrointestinal tract structure; Genetic; Glutamates; Glutamine; Gluten; Gluten-free diet; Goals; Grain; HLA-DQ2; HLA-DQ8 antigen; Image; Immune; In Vitro; Incubated; Individual; Inflammation; Inflammatory; Inflammatory Response; Ingestion; Intercept; Intervention; Intestines; Kinetics; Knock-out; Label; Lamina Propria; Life; Macroglobulins; Major Histocompatibility Complex; Malignant neoplasm of gastrointestinal tract; Measurement; Measures; Mediating; Medical; Methods; Modeling; Molecular; Morbidity - disease rate; Mus; Pathogenesis; Pathology; Pathway interactions; Patients; Peptide Transport; Peptides; Persons; Physiological; Play; Proteins; Rattus; Research; Risk; Role; Serotyping; Serum Proteins; Small Intestines; Structure; Symptoms; System; T cell response; T-Cell Activation; T-Lymphocyte; Testing; Wheat; Work; chemical reaction; cytokine; dietary; enzyme substrate; experimental study; gut inflammation; immunogenic; immunogenicity; immunoreaction; inhibitor; insight; kidney epithelial cell; milligram; novel; peptidomimetics; reconstitution; response; scaffold; small molecule; trafficking; transglutaminase 2; uptake

Project Summary Celiac disease (CeD) is a highly prevalent autoimmune disorder that occurs in response to the ingestion of wheat or related cereal grains in individuals expressing HLA-DQ2 or HLA-DQ8. CeD primarily presents in the small intestine with abdominal symptoms, but can also be associated with a myriad of extraintestinal complications and an increased risk for malignancies of the gastrointestinal tract. Despite decades of study, there are as of yet no approved medical therapies for CeD besides gluten-free diet. The life-altering symptoms, lack of treatment options, and risks that come with unmanaged disease underscore the desperate need for a deeper understanding of CeD pathogenesis. The prototypical inflammation in CeD is mediated by CD4+ T cells in response to wheat-derived gluten peptides that have been chemically modified by the enzyme transglutaminase 2 (TG2). Preliminary work in the Khosla lab has revealed that TG2 is involved in a novel gluten peptide internalization pathway involving interaction between a peptide-TG2 pair and the abundant serum protein a-2-macroglobulin (a2M). Notably, this internalization phenomenon only occurs if TG2 is bound to peptide substrate and not if TG2 is unbound or bound to non-peptide inhibitors. As a result, we believe this novel pathway may present a mechanism by which gluten peptides are internalized and subsequently presented on antigen presenting cells to CD4+ T cells. In order to explore this hypothesis, we first aim to develop a panel of novel peptidomimetic chemical inhibitors and fluorescent probes of TG2. These will enable tracking of the enzyme both throughout its catalytic cycle as well as during internalization events. We then plan to test the physiological relevance of this pathway through in vitro reconstitution with dendritic cells (DCs) and CD4+ T cells purified from CeD mice. Coculture of T cells with DCs preincubated with peptide, TG2, and a2M followed by measurement of T cell activation will allow us to determine if this mechanism contributes to the inflammatory response of CeD. Taken together, these aims seek to greater characterize the T cell response central to CeD pathogenesis. This study will provide new insight into potential targets for CeD treatment that intercept this putative gluten internalization pathway.

项目摘要 乳糜泻（CeD）是一种高度流行的自身免疫性疾病，其响应于摄入以下物质而发生： 在表达HLA-DQ 2或HLA-DQ 8的个体中的小麦或相关谷物。CeD主要存在于 小肠与腹部症状，但也可以与无数的肠外 并发症和胃肠道恶性肿瘤的风险增加。尽管经过数十年的研究， 除了无麸质饮食之外，至今还没有批准的用于CeD的医学疗法。那些改变生活的症状 缺乏治疗选择，以及未得到管理的疾病带来的风险，都强调了迫切需要 更深入地了解CeD的发病机制。 CeD中的典型炎症是由CD 4 + T细胞介导的，以响应小麦来源的谷蛋白 已被转氨酶2（TG 2）化学修饰的肽。第一阶段的初步工作 Khosla实验室揭示了TG 2参与了一种新的谷蛋白肽内化途径， 肽-TG 2对与丰富的血清蛋白α-2-巨球蛋白（α 2 M）之间的相互作用。值得注意的是， 这种内化现象仅在TG 2与肽底物结合时发生，而在TG 2未结合或 与非肽抑制剂结合。因此，我们认为这种新的途径可能提供了一种机制， 谷蛋白肽被内化，随后在抗原呈递细胞上呈递给CD 4 + T细胞。 为了探索这一假说，我们首先旨在开发一组新的拟肽化学抑制剂 和TG 2的荧光探针。这些将使得能够在整个催化循环中跟踪酶， 以及在内化事件期间。然后，我们计划通过以下方式测试该途径的生理相关性： 用从CeD小鼠纯化的树突状细胞（DC）和CD 4 + T细胞体外重建。T细胞共培养 用肽、TG 2和α 2 M预孵育DC，然后测量T细胞活化， 以确定这种机制是否有助于CeD的炎症反应。 总之，这些目标旨在更好地表征对CeD发病机制至关重要的T细胞应答。这 这项研究将为CeD治疗潜在靶点提供新的见解， 内化途径