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 腹腔疾病（CED）是一种高度普遍的自身免疫性疾病，响应于摄入 表达HLA-DQ2或HLA-DQ8的个体中的小麦或相关谷物。 CED主要提出 小肠有腹部症状，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。尽管学习了数十年， 除了无麸质饮食外，还没有CED的批准医疗疗法。改变生活的症状， 缺乏治疗方案，而不受管理的疾病带来的风险强调了迫切需要 对CED发病机理的更深入了解。 CED中的原型炎症是由CD4+ T细胞介导的，响应于小麦衍生的面筋 通过酶转谷氨酰胺酶2（TG2）化学修饰的肽。初步工作 Khosla Lab揭示了TG2参与了一种新型的麸质肽内部化途径，涉及 肽-TG2对与丰富的血清蛋白A-2-微球蛋白（A2M）之间的相互作用。尤其， 这种内在化现象仅在TG2与肽底物结合而不发生时发生，而不是TG2未结合或 与非肽抑制剂结合。结果，我们认为这种新颖的途径可能会呈现出一种机制 麸质肽被内在化，随后在抗原呈现给CD4+ T细胞的细胞上呈现。 为了探索这一假设，我们首先要开发一组新型的肽型化学抑制剂 和TG2的荧光探针。这些将使酶在整个催化循环中都可以跟踪该酶 以及在内部化事件期间。然后，我们计划通过 用树突状细胞（DC）和从CED小鼠纯化的CD4+ T细胞进行体外重构。 T细胞的共培养 DC与肽，TG2和A2M预孵育，然后测量T细胞激活将使我们 确定这种机制是否有助于CED的炎症反应。 综上所述，这些目的旨在更大的特征在于CED发病机理中心的T细胞反应。这 研究将为拦截该假定面筋的CED治疗的潜在靶标提供新的见解 内部化途径。