Illuminating B7-H3 Protein Dimerization and Function

摘要/摘要 了解免疫调节蛋白B7家族与蛋白质之间的蛋白质相互作用 它们在T细胞上的同源结合伙伴开创了癌症治疗的新纪元，检查点封锁 通过单克隆抗体抑制T细胞上的CTLA-4和PD-1，使免疫治疗发生了革命性的变化。这些 PPI提供了促进和维持T细胞反应的刺激性次级信号，它们还 导致负面的次级信号，从而下调T细胞的反应。PD-L1、PD-L2、B7-H3、B7-H4和 人类白细胞抗原-2可在非造血细胞和肿瘤上表达，但其在时间和空间上的作用 配体表达的差异及其对致病和保护性免疫反应的贡献 还没有完全阐明。许多B7家族成员，类似于其他细胞表面信号蛋白家族， 已知以二聚体的形式存在，与它们的信号状态有关。因此，B7家族相互作用的特征和 寡聚作用对于理解它们改变癌细胞的分子机制至关重要 授予致癌表型。从候选基因的方法来看，一种有趣而新颖的靶蛋白是B7- H3是B7家族的一员，在许多实体肿瘤的表面都有过表达。这项提议旨在 为了开发一种基于生物发光的顺序共振能量转移(BLI-SRET)报告PPI， 可用于定义有助于B7-H3二聚和齐聚的结构特征，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 开发一种能够在时空上区分二聚体或多聚体蛋白质复合体的BLI-SRET系统 举止。这个系统将让我们了解信号是如何通过蛋白质在分子水平上整合的 二聚和齐聚，并提供了一个平台来帮助描述这些相互作用 致病机理。在目标2中，我将检验B7-H3的特定区域对蛋白质聚集有贡献的假设 而这些相互作用的结构域的消融将扰乱蛋白质的细胞内和细胞外功能， 通过补充的免疫突触。因为每个相互作用的域可以服务于不同的下游 功能，了解调控B7-H3驱动的肿瘤发生的相似或不同机制可能会更好 实现治疗药物的路径特异性靶向，提高我们对B7-H3寡聚和 这与它的功能有何关系。这里提出的研究将支持我作为一名 独立和多产的癌症生物学家，让我对化验发展有了更好的了解， 基因编码的光学生物传感器，以及这些工具在理解复杂生物方面的应用 流程。利用皮乌尼察长期的实用专业知识和光学成像剂的开发- 和我对KRAS蛋白聚集性的详细理解，因为它与癌症的发展有关，我们 旨在开发这一系统，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B7家族致癌免疫调节蛋白的疗法。

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.