Illuminating B7-H3 Protein Dimerization and Function

总结/摘要 了解免疫调节蛋白B7家族与免疫调节蛋白之间的蛋白质-蛋白质相互作用（PPI） 它们在T细胞上的同源结合伴侣已经引领了癌症治疗的新时代， 通过单克隆抗体抑制T细胞上的CTLA-4和PD-1，彻底改变了免疫疗法。这些 PPI提供刺激性次级信号以促进和维持T细胞应答，并且它们还 有助于负次级信号，下调T细胞反应。PD-L1、PD-L2、B7-H3、B7-H4和 HHLA-2可在非造血细胞和肿瘤上表达，但其作用时间和空间上均不相同。 配体表达的差异及其对致病性和保护性免疫应答的贡献， 尚未完全阐明。许多B7家族成员，类似于细胞表面信号蛋白的其他家族， 已知以二聚体形式存在，这与它们的信号状态有关。因此，B7家族相互作用的表征和 寡聚化对于理解它们改变癌细胞的分子机制是必不可少的， 赋予致癌表型。从候选基因的方法，一个有趣的和新的靶蛋白是B7- H3是B7家族的成员，在许多实体瘤的表面过表达。这项建议旨在 开发基于生物发光的PPI序列共振能量转移（BLI-SRET）报告基因， 可用于定义有助于B7-H3二聚化和低聚化的结构特征，并确定 这些相互作用如何促进癌细胞存活和免疫逃避的机制。在目标1中，我将 开发一个BLI-SRET系统，可以区分二聚体或多聚体蛋白质复合物在时空 方式这个系统将使我们能够理解信号是如何通过蛋白质在分子水平上整合的 二聚和低聚，并提供一个平台，以帮助表征那些相互作用，有助于 发病机制在目标2中，我将检验B7-H3的特定区域有助于蛋白质聚类的假设 并且这些相互作用结构域的消融将破坏蛋白质功能，包括细胞内和细胞外， 通过互补的免疫突触由于每个相互作用的结构域可以服务于不同的下游 功能，了解调节B7-H3驱动的肿瘤发生的相似或不同机制可能更好 实现治疗剂的途径特异性靶向，并提高我们对B7-H3寡聚化的理解， 这与它的功能有什么关系。本文提出的研究将支持我的培训和发展， 独立和富有成效的癌症生物学家，使我能够更好地了解检测开发， 遗传编码光学生物传感器，以及这些工具在理解复杂生物学中的应用 流程.利用Piwnica长期以来的实践专长和光学成像剂的发展， 蠕虫小组和我对KRAS蛋白质聚类的详细理解，因为它与癌症的发展有关，我们 旨在开发这个系统，使我们能够更好地了解信号是如何在分子水平上整合的， 并利用它来帮助开发针对致癌免疫调节蛋白B7家族的治疗方法。

SUMMARY/ABSTRACT Understanding the protein-protein interactions (PPIs) between the B7-family of immune regulatory proteins and their cognate binding partners on the T-cell have led to a new era in cancer treatment, where checkpoint blockade by monoclonal antibodies inhibiting CTLA-4 and PD-1 on the T-cell has revolutionized immunotherapy. These PPIs provide both stimulatory secondary signals to promote and sustain T cell responses, and they also contribute to negative secondary signals that downregulate T cell responses. PD-L1, PD-L2, B7-H3, B7-H4 and HHLA-2 can be expressed on non-hematopoetic cells and tumors, but the role of temporal, as well as, spatial differences in ligand expression and their contributions to pathogenic and protective immune responses have not been fully elucidated. Many B7 family members, similar to other families of cell surface signaling proteins, are known to exist as dimers, related to their signaling state. Thus, characterization of B7 family interactions and oligomerization is essential to understanding the molecular mechanisms by which they alter the cancer cell to confer an oncogenic phenotype. From a candidate gene approach, an interesting and novel target protein is B7- H3, a member of the B7-family, which is overexpressed on the surface of many solid tumors. This proposal aims to develop a bioluminescence-based, sequential resonance energy transfer (BLI-SRET) reporter of PPIs that can be used to define structural features that contribute to B7-H3 dimerization and oligomerization and determine the mechanism of how these interactions promote cancer cell survival and immune evasion. In aim 1, I will develop a BLI-SRET system which can distinguish dimeric or multimeric protein complexes in a spatiotemporal manner. This system will allow us to understand how signals are integrated at a molecular level through protein dimerization and oligomerization, and provide a platform to aid characterizing those interactions that contribute to pathogenesis. In aim 2, I will test the hypothesis that defined regions of B7-H3 contribute to protein clustering and that ablation of these interacting domains will disrupt protein function, both intracellularly and extracellularly, through the complemented immune synapse. As each interacting domain may serve a different downstream function, understanding the similar or different mechanisms that regulate B7-H3-driven tumorigenesis may better enable pathway-specific targeting of therapeutics and improve our understanding of B7-H3 oligomerization and how this relates to its function. The research proposed herein will support my training and development as an independent and productive cancer biologist, allowing me to gain a greater understanding of assay development, genetically encoded optical biosensors, and the application of these tools to understand complex biological processes. Using the longstanding practical expertise and development of optical imaging agents in the Piwnica- Worms group and my detailed understanding of KRAS protein clustering as it relates to cancer development, we aim to develop this system that will allow us to better understand how signals are integrated at a molecular level, and use it to aid in developing therapeutics to target the B7-family of oncogenic immune regulatory proteins.