Deciphering biased agonistic activation of mu-opioid receptor by novel optogenetic hydrogen peroxide sensor

新型光遗传学过氧化氢传感器破译μ阿片受体的偏向激动激活

基本信息

  • 批准号:
    10604662
  • 负责人:
  • 金额:
    $ 4.16万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2023
  • 资助国家:
    美国
  • 起止时间:
    2023-03-01 至 2025-02-28
  • 项目状态:
    未结题

项目摘要

ABSTRACT: In 2017 the U.S. Department of Health and Human Services declared the ongoing opioid epidemic a public health crisis and more than 100,000 people died due to opioid overdose in 2021. A critical part of the solution is to understand the fundamental reaction and adaptation of brain circuits to stimulation by opioids. For example, the desensitization of opioid receptors is a critical problem in pain management because it requires increasing doses of analgesic compounds, which could contribute to developing a drug addiction. Recently, it has been shown that the activation of mu and kappa opioid receptors in neurons causes the production of reactive oxygen species (ROS) through a pathway involving NADPH oxidase and c-Jun N-terminal kinase. Therefore, this distinct response, downstream from the receptor, could be utilized to detect specific opioid receptor activation and modulation. However, we currently lack sensitive fluorescent sensors, which would allow us to directly monitor pathways downstream from mu-opioid receptor (MOR) activation in real-time. Current limitations of contemporary sensors are slow response time, low specificity, low signal output, low brightness, and toxicity. My goal is to develop a genetically encoded sensor protein that detects ROS levels at endogenous levels with response time and signal amplitudes that will enable monitoring of neuronal systems upon MOR activation in brain tissue. I have developed a novel fluorescent ROS sensor with significantly improved signaling amplitude, sensitivity, and response kinetics compared to previous sensors. I used a newly identified insertion site on OxyR, a bacterial hydrogen peroxide binding protein, that putatively improved allosteric coupling to the fluorescent reporter domain. We will increase the fidelity of this tool with new green fluorescent protein (AausFP1) that exhibits exceptional quantum yield and brightness. I will optimize a new ROS sensor at an unprecedented rate through a multifaceted approach that combines rational, computational, and evolutionary protein engineering. My objective is to express this novel tool in MOR positive neurons and to link ROS signals to MOR activity pharmacologically. I hypothesize that ROS signals in MOR neurons will increase under morphine but not fentanyl through a pathway including c-Jun N-terminal Kinase, Peroxiredoxin 6 and NADPH oxidase. Furthermore, I hypothesize that we will observe a decrease in ROS transients under repeated drug exposure reflecting the desensitization of MORs. At the end, I will have a novel and highly specific sensor for monitoring opioid receptor activity and adaptivity. My proposal is significant because, for the first time, we will be able to monitor the adaptation of this clinically relevant signaling pathway to opioid exposure. My approach is innovative because it combines novel protein engineering and monitoring of opioid-triggered signals to dissect a difficult-to-access neuronal signaling pathway. Furthermore, this approach could be broadly applied in future studies to monitor the activity of opioid receptors during drug exposure and link the subsequent changes in neuronal signaling and plasticity to motivated behaviors, or analgesic tolerance.
摘要:2017年,美国卫生与公众服务部宣布阿片类药物正在流行 2021年,一场公共卫生危机和10万多人死于阿片类药物过量。一个关键的部分, 解决办法是了解大脑回路对阿片类药物刺激的基本反应和适应。为 例如,阿片受体的脱敏是疼痛管理中的一个关键问题,因为它需要 增加止痛化合物的剂量,这可能有助于形成毒瘾。最近,它 已经证明,神经元中的u和kappa阿片受体的激活导致产生 活性氧物种(ROS)通过NADPH氧化酶和c-jun氨基末端激酶途径被激活。 因此,受体下游的这种不同的反应可以用来检测特定的阿片类药物 受体的激活和调节。然而,我们目前缺乏灵敏的荧光传感器,这将允许 美国将直接实时监测阿片受体(MOR)激活的下游通路。当前 当代传感器的局限性是响应时间慢、特异性低、信号输出低、亮度低、 和毒性。我的目标是开发一种基因编码的传感器蛋白,它可以检测ROS水平 内源性水平,具有响应时间和信号幅度,将使神经元监测成为可能 系统对脑组织中MOR的激活。我已经开发了一种新型的荧光ROS传感器 与以前的传感器相比,显著提高了信号幅度、灵敏度和响应动力学。我 在细菌过氧化氢结合蛋白OxyR上使用了新发现的插入位点,推测 改进了与荧光报告结构域的变构偶联。我们将通过新的工具来提高此工具的保真度 绿色荧光蛋白(AousFP1),表现出非凡的量子产率和亮度。我将优化一个 新的ROS传感器以前所未有的速度通过多方面的方法结合了Rational、 计算和进化蛋白质工程。我的目标是用MOR Positive来表达这个新工具 并将ROS信号与MOR的药理活性联系起来。我假设ROS在MOR中发出信号 在吗啡作用下,神经元会增加,但芬太尼不会通过包括c-jun氨基末端激酶在内的途径增加神经元。 过氧化还蛋白6和NADPH氧化酶。此外,我假设我们将观察到ROS的下降 反复药物暴露下的短暂反应反映了MORS的脱敏。最后,我会有一本小说 以及用于监测阿片受体活性和适应性的高度特异性传感器。我的建议意义重大 因为,我们将第一次能够监测这一临床相关信号通路的适应性。 与阿片类药物接触有关。我的方法是创新的,因为它结合了新的蛋白质工程和监测 阿片类药物触发的信号来解剖一条难以进入的神经信号通路。此外,这种方法 可广泛应用于未来的研究,以监测阿片受体的活性在药物暴露和 将随后神经元信号和可塑性的变化与动机行为或止痛耐受联系起来。

项目成果

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Justin Lee其他文献

Justin Lee的其他文献

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{{ truncateString('Justin Lee', 18)}}的其他基金

Ultrasound-Controlled Immunotherapy for Targeted Treatment of Solid Tumors
超声控制免疫疗法用于实体瘤的靶向治疗
  • 批准号:
    10399420
  • 财政年份:
    2021
  • 资助金额:
    $ 4.16万
  • 项目类别:

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