A novel small-molecule approach to detect specifc peptide-MHC complexes

描述（由申请人提供）：抗原呈递开始于抗原呈递细胞（apc）内小肽和主要组织相容性复合体（MHC）的结合。识别特定的非自身肽- mhc复合物是一个人抵抗感染能力的核心，而错误地将自身肽- mhc复合物识别为异物是导致自身免疫病理的原因，如在1型糖尿病中发现的。目前的自身免疫疗法不具有特异性，因此由于普遍免疫抑制的副作用而变得复杂。毫无疑问，更好地了解这两个过程的差异将导致更具体的治疗方法。肽- mhc复合物形成的细胞器、引导其运输到细胞表面的过程以及控制其寿命的机制都是未知的，因为迄今为止还不可能以高分辨率可视化活细胞中肽- mhc复合物的形成。本研究采用了一种新开发的技术——二部四胱氨酸显示——来成像和跟踪活细胞中肽- mhc复合物的形成。在二部四半胱氨酸展示中，两个结合伙伴上的半胱氨酸对在复合体形成时紧密并列。这个新形成的四胱氨酸基序被FIAsH和ReAsH快速特异性标记。对于这个提议，我们将首先（AIM 1），设计肽- mhc复合物用于分裂四胱氨酸展示，并评估它们在复合物中结合FIAsH和ReAsH的能力，而不是单独结合。然后，我们将（AIM 2）分析这些蛋白在活细胞中的表达，并跟踪肽- mhc复合物形成的时空调节。最后（AIM 3），我们将利用在活细胞中跟踪抗原呈递的能力来筛选小分子和肽配体库，以调节抗原呈递的能力。可视化和跟踪肽- mhc复合物的能力有可能解决有关抗原呈递的生物学机制的问题。最终，阻断负责自身免疫的复合物形成的能力可能是治疗自身免疫的一种更具体的方法。与使用荧光蛋白或抗体相比，拆分四胱氨酸显示实现了空间和时间分辨率。该提案的重点是开发能够特异性检测蛋白质和蛋白质复合物的成像工具，而不使用荧光蛋白或抗体，通过使用“前荧光”小分子作为特定化学结构的“开启”荧光传感器。这些研究将提高我们对正常和疾病生理学的理解，并指导新疗法的发展。

DESCRIPTION (provided by applicant): Antigen presentation begins with the association of small peptides and the major histocompatability complex (MHC) within antigen presenting cells (APCs). Recognition of specific non-self peptide-MHC complexes is central to one's ability to fight infection, while mistakenly recognizing self-peptide-MHC complexes as foreign is responsible for autoimmune pathology such as is found in type-1 diabetes. Current autoimmune therapies are not specific, and thus complicated by the side-effects of general immune suppression. There is little doubt that a better understanding of the differences in these two processes will lead to more specific therapies. The cellular organelle in which peptide-MHC complexes form, the processes guiding their transport to the cell surface, and the mechanisms controlling their longevity are all unknown because it has heretofore been impossible to visualize the formation of peptide-MHC complexes in live cells with high resolution. This proposal applies a newly developed technique - bipartite tetracysteine display - to image and track the formation of peptide-MHC complexes in live cells. In bipartite tetracysteine display, pairs of cysteines on each of two binding partners become closely juxtaposed upon complex formation. This newly formed tetracysteine motif is rapidly and specifically labeled by FIAsH and ReAsH. For this proposal, we will first (AIM 1), engineer peptide-MHC complexes for split tetracysteine display and evaluate their ability to bind FIAsH and ReAsH in complex but not separately. We will then (AIM 2), analyze expression of these proteins in live cells and track the spatial and temporal regulation of peptide-MHC complex formation. Finally (AIM 3), we will exploit the ability to track antigen presentation in live cells to screen a library of small molecule and peptide ligands for the ability to modulate antigen presentation. The ability to visualize and track peptide-MHC complexes has the potential to address questions concerning the biological mechanism of antigen presentation. Eventually, the ability to block the formation of complexes responsible for autoimmunity could be a more specific approach to the treatment of autoimmunity. PUBLIC HEALTH RELEVANCE Split tetracysteine display achieves spatial and temporal resolution over what can be achieved using fluorescent proteins or antibodies. This proposal focuses on the development of imaging tools capable of specifically detecting proteins and protein complexes without the use fluorescent proteins or antibodies through the use of 'pro-fluorescent' small molecules that act as 'turn-on' fluorescent sensors for particular chemical structures. These studies will improve our understanding of normal and disease physiology and guide the development of new therapies.