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

描述(由申请人提供)：抗原呈递始于小肽与抗原呈递细胞(APC)内的主要组织相容性复合体(MHC)的关联。识别特定的非自体多肽-MHC复合体是一个人抗感染能力的核心，而错误地将自体多肽-MHC复合体识别为外源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没有特效性，因此普遍免疫抑制的副作用使其复杂化。毫无疑问，更好地了解这两个过程中的差异将导致更具体的治疗方法。多肽-MHC复合体形成的细胞器，引导它们运输到细胞表面的过程，以及控制它们寿命的机制都是未知的，因为到目前为止，还不可能以高分辨率在活细胞中可视化多肽-MHC复合体的形成。这项建议应用了一种新开发的技术--两部分四半胱氨酸显示--来成像和跟踪活细胞中多肽-MHC复合体的形成。在两部分四半胱氨酸展示中，两个结合伙伴的每一个上的半胱氨酸对在形成络合物时紧密并列。这个新形成的四半胱氨酸基序被FIAsH和ReAsH快速和特异地标记。对于这项建议，我们将首先(目标1)，设计多肽-MHC复合体用于四半胱氨酸的裂解展示，并评估它们在复合体中结合FIAsH和ReAsH的能力，而不是单独结合。然后，我们将(目标2)，分析这些蛋白质在活细胞中的表达，并跟踪多肽-MHC复合体形成的时空调节。最后(目标3)，我们将利用在活细胞中跟踪抗原提呈的能力来筛选小分子和多肽配体的库，以获得调节抗原提呈的能力。可视化和跟踪多肽-MHC复合体的能力有可能解决与抗原呈递的生物学机制有关的问题。最终，阻止与自身免疫有关的复合体的形成的能力可能是治疗自身免疫的更具体的方法。与公共健康相关的分离式四半胱氨酸显示器实现了对使用荧光蛋白或抗体可以实现的空间和时间分辨率。这项提议的重点是开发成像工具，该工具能够专门检测蛋白质和蛋白质复合体，而不使用荧光蛋白质或抗体，方法是使用“亲荧光”小分子，这些小分子充当特定化学结构的“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，并指导新疗法的开发。

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.