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

项目摘要 乳糜泻(CED)是一种高度流行的自身免疫性疾病，发生于摄入 表达人类白细胞抗原DQ2或人类白细胞抗原DQ8的个体的小麦或相关谷物。CED主要出现在 小肠有腹部症状，但也可伴有无数的肠外症状 并发症和胃肠道恶性肿瘤的风险增加。尽管进行了数十年的研究， 到目前为止，除了无麸质饮食外，还没有批准的治疗CED的药物。改变生活的症状， 缺乏治疗选择，以及未得到控制的疾病带来的风险，突显了对 加深对CED发病机制的认识。 CED的典型炎症是由CD4T细胞对小麦面筋蛋白的反应所介导的 已被转谷氨酰胺酶2(TG2)化学修饰的多肽。前期工作： Khosla实验室揭示了TG2参与了一种新的谷蛋白多肽内化途径，包括 多肽-TG2对与丰富的血清蛋白a-2-巨球蛋白(A2M)的相互作用。值得注意的是， 这种内化现象只有在TG2与多肽底物结合时才会发生，而不是在TG2未结合或 与非肽类抑制剂结合。因此，我们认为这种新的途径可能提供了一种机制，通过它 面筋多肽被内化，随后在抗原提呈细胞上呈递给CD4T细胞。 为了探索这一假设，我们首先致力于开发一组新型的模拟多肽的化学抑制剂。 和TG2的荧光探针。这些将使跟踪酶在其整个催化循环作为 以及在内部化事件期间。然后，我们计划通过以下方式测试这条通路的生理学相关性 从CED小鼠纯化的树突状细胞(DC)和CD4T细胞的体外重建。T细胞的共培养 将树突状细胞与多肽、TG2和a2M预先孵育，然后测量T细胞激活将允许我们 以确定该机制是否参与了CED的炎症反应。 综上所述，这些目标试图更好地表征CED发病机制中的核心T细胞反应。这 这项研究将为CED治疗截留这种假定面筋的潜在靶点提供新的见解 内化途径。

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.