Dissecting protein kinase A regulation of neurons using synthetic approaches

使用合成方法剖析蛋白激酶 A 对神经元的调节

• 批准号: BB/X008215/1
• 负责人: Matthew Gold
• 金额: $ 63.04万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX008215%2F1
• 关键词: Dissecting; protein; kinase; regulation; neurons

Cells throughout the body present surface receptors that enable them to respond to external stimuli such as hormones and neurotransmitters. A common signal transduction mechanism is for these external primary messengers to trigger accumulation of the 'second messenger' cyclic AMP (cAMP) within cells. The major receptor for cAMP - protein kinase A (PKA) - responds to cAMP elevations to bring about physiological changes by phosphorylating proteins. The myriad processes controlled by PKA phosphorylation include sympathetic stimulation of heart rate, control of water reuptake in the kidney, and control of the excitability and shape of brain cells called neurons. Research in recent years has revealed that cAMP signalling in cells is organised in 'nanodomains' sometimes with a diameter of less than 100 nanometres. The precise location of a copy of PKA in a cell therefore dictates whether it will be activated by a given stimulus. Anchoring proteins position PKA at different sub-cellular locations, and these anchoring proteins are thought to direct the kinase to phosphorylate different sub-sets of substrates linked to different functions. Furthermore, targeting of individual PKA anchoring sites is considered a promising strategy for selective disruption of pathological processes supported by PKA phosphorylation such as neuronal excitability underlying epilepsy. However, fundamental aspects of our current understanding of PKA anchoring have not been resolved, and the precise role that different PKA anchoring proteins play in neuronal excitability is yet to be disentangled. These areas would benefit from new technologies for manipulating PKA activity in time and space.In this study, we will develop two innovative technologies inspired by the field of synthetic biology that may be applied to direct PKA to specific anchoring proteins, and to dictate when the kinase is activated under the control of blue light. We will then utilise these technologies in combination with existing methods to investigate fundamentals of PKA signalling in nanodomains, and the specific roles of different PKA anchoring proteins in controlling the shape and excitability of neurons. To enable specific anchoring of PKA to individual anchoring proteins, we will take advantage of protein domains that enable molecular 'gluing' of proteins in living cells. This work will involve the development of two cell lines using gene editing technologies. To develop a photo-activatable form of PKA, we will perform high-throughput screening with a library of PKA regulatory and catalytic subunits in which the elements that normally respond to cAMP are replaced with ones that respond to blue light. The most promising combinations will be optimised and validated using protein binding and activity assays. Our investigations of cAMP nanodomain fundamentals will include determining how individual PKA-anchoring protein complexes respond to different primary stimuli using targeted fluorescent reporters of PKA activity and quantitative proteomics. The final component of our study will focus on clarifying how changes in cAMP and PKA are linked to epilepsy using a slice model preparation. We will also measure changes in excitability and morphology in cultured neurons to determine how different PKA anchoring sites control these aspects of neuronal function.We have assembled a team of investigators with complementary expertise in techniques ranging from protein engineering to electrophysiology, and in fields including cAMP signalling and epilepsy. The proposed research will benefit from collaboration with experts in photoactivation and quantitative proteomics. In addition to advancing fundamental knowledge of nanodomain cAMP signalling in neurons, the new technologies developed during this research will benefit researchers focusing on the many other roles played by PKA throughout the body.

全身的细胞都存在表面受体，使它们能够对激素和神经递质等外部刺激做出反应。常见的信号转导机制是这些外部主要信使触发细胞内“第二信使”环磷酸腺苷 (cAMP) 的积累。 cAMP 的主要受体 - 蛋白激酶 A (PKA) - 对 cAMP 升高做出反应，通过磷酸化蛋白质带来生理变化。 PKA 磷酸化控制的众多过程包括心率的交感神经刺激、肾脏水再摄取的控制以及称为神经元的脑细胞的兴奋性和形状的控制。近年来的研究表明，细胞中的 cAMP 信号传导以“纳米域”的形式组织，有时直径小于 100 纳米。因此，细胞中 PKA 副本的精确位置决定了它是否会被给定的刺激激活。锚定蛋白将 PKA 定位在不同的亚细胞位置，并且这些锚定蛋白被认为指导激酶磷酸化与不同功能相关的不同底物子集。此外，靶向单个 PKA 锚定位点被认为是选择性破坏 PKA 磷酸化支持的病理过程（例如癫痫背后的神经元兴奋性）的有前途的策略。然而，我们目前对 PKA 锚定的理解的基本方面尚未得到解决，不同的 PKA 锚定蛋白在神经元兴奋性中所起的精确作用也尚未阐明。这些领域将受益于在时间和空间上操纵 PKA 活性的新技术。在这项研究中，我们将开发两项受合成生物学领域启发的创新技术，这些技术可用于将 PKA 导向特定的锚定蛋白，并指示何时激酶在蓝光的控制下被激活。然后，我们将利用这些技术与现有方法相结合，研究纳米域中 PKA 信号传导的基础原理，以及不同 PKA 锚定蛋白在控制神经元形状和兴奋性中的具体作用。为了使 PKA 能够特异性锚定到单个锚定蛋白上，我们将利用能够在活细胞中对蛋白质进行分子“粘合”的蛋白质结构域。这项工作将涉及使用基因编辑技术开发两种细胞系。为了开发光激活形式的 PKA，我们将使用 PKA 调节和催化亚基库进行高通量筛选，其中通常响应 cAMP 的元件被替换为响应蓝光的元件。最有希望的组合将使用蛋白质结合和活性测定进行优化和验证。我们对 cAMP 纳米结构域基础的研究将包括使用 PKA 活性的靶向荧光报告基因和定量蛋白质组学来确定各个 PKA 锚定蛋白复合物如何响应不同的初级刺激。我们研究的最后一部分将侧重于使用切片模型制备来阐明 cAMP 和 PKA 的变化如何与癫痫相关。我们还将测量培养神经元的兴奋性和形态的变化，以确定不同的 PKA 锚定位点如何控制神经元功能的这些方面。我们组建了一支研究人员团队，他们在从蛋白质工程到电生理学的技术以及包括 cAMP 在内的领域具有互补的专业知识信号传导和癫痫。拟议的研究将受益于与光活化和定量蛋白质组学专家的合作。除了增进神经元纳米结构域 cAMP 信号传导的基础知识之外，本研究中开发的新技术还将有利于研究人员关注 PKA 在整个身体中发挥的许多其他作用。