Live Cell Isoform-specific Akt Analyses

活细胞亚型特异性 Akt 分析

• 批准号: 10796490
• 负责人: Degeng Wang
• 金额: $ 47.53万
• 依托单位: TEXAS TECH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-10 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10796490
• 关键词: AKT2 gene; Adenine; Apoptosis; Binding; Biological Assay; Bypass; CRISPR/Cas technology; Cells; Communities; Development; Diploidy; Disease; Drug Targeting; Dyes; Ectopic Expression; Educational Activities; Fluorescence; G Cells; G-substrate; Gatekeeping; Generations; Genes; Genomics; Goals; Growth; HCT116 Cells; Homologous Protein; Human; Human Genome; In Vitro; Knock-in Mouse; Knockout Mice; Metabolism; Methods; Mitotic Cell Cycle; Modeling; Mutate; Mutation; PIK3CG gene; PTEN gene; Phosphotransferases; Process; Protein Isoforms; Protein Kinase; Reaction; Research; Research Activity; Research Personnel; Sequence Homology; Signal Transduction; Site; Students; Substrate Specificity; System; Testing; Transfection; Western Blotting; analog; career; chemical genetics; diabetic; drug development; expression vector; fluorescence microscope; genome editing; high throughput screening; human disease; improved; interest; kinase inhibitor; mutant; nanoparticle; nanoparticle delivery; preference; prime editing; repaired; screening; therapeutic development; therapeutic target; thiophosphate; undergraduate student

This project is developing live-cell kinase analysis methods that can distinguish close isoforms. Akt1 and Akt2 are our initial kinase model. The two kinases share high sequence homology, but mysteriously display functional difference in development, diabetics and et al. We aim to develop fluorescent live-cell isoform- specific Akt activity quantification, and to determine whether different Akt1 and Akt2 substrate specificity and/or preference are an underlying mechanism. The methods are widely applicable. The project will nurture the growth of many undergraduate researchers to their next stage of their biomedical career. By combining the well-established chemical-genetic method developed by Dr. Kevan Shokat and nanoparticle-delivery of the cell-impermeable bulky analog N6-benzyl-ATP-g-S (A*TP-g-S), we achieved specific substrate tagging in live cells, that is, the ability to distinguish the kinase reaction of interest from those of the > 500 kinases encoded in the human genome – a prerequisite for live-cell “kinase assay”. Briefly, according to the Shokat method, Akt1/2 gatekeeper Met (M) was mutated to Gly (G) to enlarge the ATP binding pockets to accommodate A*TP; i.e., creation of the Akt1M-G and Akt2M-G mutants. We showed that only Akt1/2M-G could efficiently use delivered A*TP-g-S due to the bulkier adenine moiety, and thiophosphate- tag their substrates. Currently, A*TP is being tagged with the MANT or TNP fluorogenic dyes. Exclusive Akt1/2M-G binding to nanoparticle-delivered MANT-/TNP-A*TP will activate their fluorescence, which in turn quantify Akt1/2 ATP-binding, and thus enzymatic, activities in intact live cells; that is, live-cell quantification of Akt1/2 kinase activities. We are also generating the mutations via genome editing, so that Akt1/2M-G genes stay in native genomic context. Our aims to accomplish the goals are: Aim 1: Fluorogenic live-cell quantification of Akt ATP binding activities via expression vectors; Aim 2: Adapting the method to prime-edited HCT116 human cells with enlarged Akt1 or Akt2 ATP binding pockets, i.e., the Akt1M→G cells and the Akt2M→ G cells; and Aim 3: To identify and differentiate the Akt1 and Akt2 spectrums of substrates. Significance: We are developing a widely applicable system for live-cell kinase studies. Kinase-substrate relationship analysis via substrate tagging in live cells will be established. Our fluorogenic live-cell kinase assay will bypass usage of artificial substrates to enable, among others, distinguishing homologous kinase isoforms such as Akt1 and Akt2. The results will set a new and general paradigm for studying protein kinases. Akt1 and Akt2 have long been targeted for drug and therapeutic development. Identification of their respective spectrum of substrates will help guide further development and improvement efforts for relevant human diseases. Additionally, the project provides an excellent interdisciplinary platform for students to integrate classroom education and research activities.

该项目正在开发可以区分相近异构体的活细胞激酶分析方法。 Akt1 和 Akt2 是我们最初的激酶模型。这两种激酶具有高度的序列同源性，但神秘地显示 发育、糖尿病等方面的功能差异。我们的目标是开发荧光活细胞亚型- 特异性 Akt 活性定量，并确定 Akt1 和 Akt2 底物特异性是否不同 和/或偏好是一个基本机制。这些方法应用广泛。该项目将培育 许多本科研究人员成长到生物医学职业的下一阶段。 通过结合 Kevan Shokat 博士开发的成熟化学遗传方法和 细胞不可渗透的大体积类似物 N6-苄基-ATP-g-S (A*TP-g-S) 的纳米粒子递送，我们实现了 活细胞中的特异性底物标记，即区分感兴趣的激酶反应的能力 人类基因组中编码的超过 500 种激酶的那些——这是活细胞“激酶测定”的先决条件。简要地， 根据 Shokat 方法，Akt1/2 Gatekeeper Met (M) 突变为 Gly (G) 以放大 ATP 装订口袋可容纳 A*TP；即创建 Akt1M-G 和 Akt2M-G 突变体。我们证明了 由于腺嘌呤部分较大，只有 Akt1/2M-G 才能有效地使用所传递的 A*TP-g-S，并且硫代磷酸盐- 标记它们的底物。目前，A*TP 正在使用 MANT 或 TNP 荧光染料进行标记。独家的 Akt1/2M-G 与纳米颗粒传递的 MANT-/TNP-A*TP 结合将激活它们的荧光，进而激活它们的荧光 量化完整活细胞中的 Akt1/2 ATP 结合活性和酶活性；也就是说，活细胞定量 Akt1/2 激酶活性。我们还通过基因组编辑生成突变，以便 Akt1/2M-G 基因 留在本地基因组环境中。我们实现这些目标的目标是： 目标 1：荧光活细胞 通过表达载体量化 Akt ATP 结合活性；目标 2：使方法适应素编辑 具有扩大的 Akt1 或 Akt2 ATP 结合袋的 HCT116 人类细胞，即 Akt1M→G 细胞和 Akt2M→ G细胞；目标 3：识别和区分底物的 Akt1 和 Akt2 光谱。 意义：我们正在开发一种广泛适用的系统，用于活细胞激酶研究。激酶底物 将通过活细胞中的底物标记进行关系分析。我们的荧光活细胞激酶 检测将绕过人工底物的使用，从而能够区分同源激酶等 同种型，例如 Akt1 和 Akt2。这些结果将为研究蛋白激酶建立一个新的通用范例。 Akt1 和 Akt2 长期以来一直是药物和治疗开发的目标。各自的身份识别 底物谱将有助于指导相关人类的进一步开发和改进工作 疾病。此外，该项目为学生提供了一个优秀的跨学科平台 课堂教育和研究活动。