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.

该项目正在开发活细胞激酶分析方法，可以区分密切的亚型。akt 1和 Akt 2是我们的初始激酶模型。这两种激酶具有高度的序列同源性，但神秘地显示， 在发育、糖尿病等方面的功能差异。我们的目标是开发荧光活细胞异构体， 特异性Akt活性定量，并确定是否存在不同的Akt 1和Akt 2底物特异性 和/或偏好是一种潜在的机制。这些方法具有广泛的适用性。该项目将培养 许多本科生研究人员的成长，他们的生物医学职业生涯的下一个阶段。 通过结合Kevan Shokat博士开发的成熟的化学遗传方法， 纳米颗粒递送细胞不可渗透的庞大类似物N6-苄基-ATP-g-S（A*TP-g-S），我们实现了 活细胞中特异性底物标记，即区分感兴趣的激酶反应与 人类基因组中编码的> 500种激酶中的那些-活细胞“激酶测定”的先决条件。简言之， 根据Shokat方法，将Akt 1/2看门人Met（M）突变为Gly（G）以扩增ATP 结合袋以容纳A*TP;即，Akt 1 M-G和Akt 2 M-G突变体的产生。我们发现 只有Akt 1/2 M-G可以有效地利用递送的A*TP-g-S，这是由于较大的腺嘌呤部分和硫代磷酸- 标记它们的底物。目前，A*TP被标记有MANT或TNP荧光染料。独家 Akt 1/2 M-G与纳米颗粒递送的MANT-/TNP-A*TP结合将激活它们的荧光，这反过来 在完整的活细胞中定量Akt 1/2 ATP结合，从而定量酶活性;即， Akt 1/2激酶活性。我们还通过基因组编辑产生突变，这样Akt 1/2 M-G基因 保持在天然基因组环境中。我们的目标是：目标1：荧光活细胞 通过表达载体定量Akt ATP结合活性;目的2：使该方法适应引物编辑的 具有增大的Akt 1或Akt 2 ATP结合口袋的HCT 116人细胞，即，Akt 1 M →G细胞和Akt 2 M → 目的3：鉴定和区分底物的Akt 1和Akt 2谱。 意义：我们正在开发一个广泛适用于活细胞激酶研究的系统。激酶底物 将建立通过活细胞中的底物标记的关系分析。我们的荧光活细胞激酶 测定将绕过人工底物的使用，以使得能够区分同源激酶 Akt 1和Akt 2等亚型。这一结果将为蛋白激酶的研究提供一个新的、通用的范式。 Akt 1和Akt 2长期以来一直是药物和治疗开发的目标。识别各自的 底物谱将有助于指导相关人类 疾病此外，该项目为学生提供了一个优秀的跨学科平台来整合 课堂教学和研究活动。