Illuminating B7-H3 Protein Dimerization and Function

阐明 B7-H3 蛋白二聚化和功能

• 批准号: 10179335
• 负责人: Margie Nicole Sutton
• 金额: $ 7.11万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10179335
• 关键词: Ablation; Alleles; Amino Acids; Attenuated; Binding; Binding Proteins; Biochemical; Biological Assay; Biological Process; Biological Response Modifiers; Bioluminescence; Biosensor; Bladder; CD276 gene; CD28 gene; CTLA4 gene; Candidate Disease Gene; Cell Surface Proteins; Cell Survival; Cell membrane; Cell surface; Cells; Colorectal; Complement; Complex; Development; Diffuse; Dimerization; EGF gene; Energy Transfer; Enterobacteria phage P1 Cre recombinase; Epidermal Growth Factor Receptor; Family; Family member; Fluorescence; Homeostasis; Immune; Immune Evasion; Immune response; Immunomodulators; Immunotherapy; Integral Membrane Protein; KRAS2 gene; Kidney; Kinetics; Ligands; Luciferases; Lung; Lymphocyte; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of prostate; Methods; Molecular; Monitor; Monoclonal Antibodies; Oncogenic; Optics; Ovarian; Pancreas; Pathogenesis; Pathogenicity; Pathway interactions; Phenotype; Phosphotransferases; Physiological; Property; Protein Analysis; Protein Family; Proteins; Receptor Cell; Reporter; Research; Roentgen Rays; Role; Signal Transduction; Signaling Protein; Solid Neoplasm; Structure; Structure-Activity Relationship; Surface; System; T cell response; T-Lymphocyte; Techniques; Technology; Testing; Therapeutic; Time; Training; VTCN1 gene; Work; assay development; base; cancer cell; cancer therapy; differential expression; dimer; extracellular; genetic regulatory protein; imaging agent; immune checkpoint; immune checkpoint blockade; immunological synapse; immunoregulation; improved; in vivo; inhibiting antibody; innovation; interest; luminescence; melanoma; member; mutant; novel; novel strategies; optical imaging; overexpression; programmed cell death ligand 1; programmed cell death protein 1; protein complex; protein function; protein protein interaction; receptor; recombinase-mediated cassette exchange; response; spatiotemporal; targeted treatment; tool; tumor; tumorigenesis

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.

摘要/摘要 了解免疫调节蛋白 B7 家族之间的蛋白质-蛋白质相互作用 (PPI) 它们在 T 细胞上的同源结合伙伴开创了癌症治疗的新时代，其中检查点封锁 通过抑制 T 细胞上的 CTLA-4 和 PD-1 的单克隆抗体彻底改变了免疫疗法。这些 PPI 提供刺激性次级信号来促进和维持 T 细胞反应，而且它们还 导致负次级信号下调 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，但其作用随时间和空间的变化而变化。 配体表达的差异及其对致病性和保护性免疫反应的贡献 尚未完全阐明。许多 B7 家族成员，与细胞表面信号蛋白的其他家族类似， 已知以二聚体形式存在，与其信号传导状态有关。因此，B7 家族相互作用的特征和 寡聚化对于理解它们改变癌细胞的分子机制至关重要 赋予致癌表型。从候选基因方法来看，一个有趣且新颖的靶蛋白是 B7- H3 是 B7 家族的成员，在许多实体瘤表面过度表达。该提案旨在 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 二聚和寡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，我会 开发 BLI-SRET 系统，可以在时空范围内区分二聚体或多聚体蛋白质复合物 方式。该系统将使我们能够了解信号如何通过蛋白质在分子水平上整合 二聚化和寡聚化，并提供一个平台来帮助表征那些有助于 至发病机制。在目标 2 中，我将检验 B7-H3 的定义区域有助于蛋白质聚类的假设 消除这些相互作用的结构域会破坏细胞内和细胞外的蛋白质功能， 通过互补的免疫突触。由于每个交互域可能服务于不同的下游 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会更好 实现治疗的通路特异性靶向并提高我们对 B7-H3 寡聚化的理解 这与其功能有何关系。本文提出的研究将支持我作为一名 独立且富有成效的癌症生物学家，使我能够更好地了解检测开发， 基因编码的光学生物传感器，以及这些工具的应用来理解复杂的生物 流程。利用 Piwnica 长期的实践专业知识和光学显像剂的开发 蠕虫小组和我对 KRAS 蛋白聚类的详细了解，因为它与癌症发展有关，我们 旨在开发这个系统，使我们能够更好地了解信号如何在分子水平上整合， 并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。