Optogenetic protein design for hypertrophic signaling pathways

光遗传学主要使用光门离子通道(来自低等生物)来触发或抑制 神经动作电位。心血管领域的类似研究使用了这些光响应 调节收缩和起搏的外来蛋白质。然而，光门离子通道不能询问 控制行为和动态平衡的细胞内机制。因此，我们开发了一种 创造内源蛋白质的光响应类似物的蛋白质工程策略。建议数 研究计划的重点是设计、构建和验证蛋白质的光遗传信号 有助于心肌内稳态，应用这项技术来解开 导致心肌肥大和纤维化。目标1：光基因工程获取生化 肥大和纤维化的途径。我们将采用一种新的光基因工程策略来创造 光反应信号级联定位于特定的细胞内位置，重点是cAMP介导的 信号通路。目标2：光引导下绘制介导肥大的信号结构域。这个 目标1中概述的光遗传结构将用于探索不同信号事件的影响 细胞内位置对心肌细胞行为的影响，重点是肥大相关的反应。目标3： 介导纤维化的细胞间通讯的光导图谱。光遗传学类似物 内源性蛋白质将被用来评估两种不同类型的心肌细胞之间的细胞间串扰。光- 工程信号通路的触发激活提供了梳理协作 在这些不同的细胞群体之间观察到的信号网络。

Optogenetics has primarily employed light gated ion channels (derived from lower organisms) to trigger or inhibit the neuronal action potential. Analogous studies in the cardiovascular field have used these photoresponsive foreign proteins to regulate contractility and pacing. However, light gated ion channels are unable to interrogate the intracellular mechanisms that control behavior and homeostasis. As a consequence, we have developed a protein engineering strategy for creating light responsive analogs of endogenous proteins. The proposed research program is focused on the design, construction, and validation of optogenetic signaling for proteins that contribute to myocardial homeostasis, and the application of this technology to unravel the mechanisms that drive cardiac hypertrophy and fibrosis. Aim 1: Optogenetic Engineering to Access the Biochemical Pathways of Hypertrophy and Fibrosis. We will employ a novel optogenetic engineering strategy to create light responsive signaling cascades localized at specific intracellular sites, with a focus on cAMP-mediated signaling pathways. Aim 2: Light Guided Mapping of the Signaling Domains that Mediate Hypertrophy. The optogenetic constructs outlined in Aim 1 will be used to explore the impact of signaling events at distinct intracellular locations on cardiomyocyte behavior, with an emphasis on hypertrophic relevant responses. Aim 3: Light Guided Mapping of the Intercellular Communication that Mediates Fibrosis. Optogenetic analogs of endogenous proteins will be used to assess intercellular crosstalk between two distinct cardiac cell types. Light- triggered activation of engineered signaling pathways offers the opportunity to tease apart a collaborative signaling network that has been observed between these distinct cell populations.