Emergent cellular functions of GPCRs and myosins

项目总结 细胞过程，如信号和膜交通，来自一系列动态的、瞬时的 拥挤的细胞环境中的蛋白质-蛋白质相互作用。异常的蛋白质-蛋白质相互作用 经常与衰弱或致命的疾病有关，如糖尿病、神经退行性疾病和 癌症。已建立的结构和细胞生物学技术大多局限于解剖细胞的功能 稳定的蛋白质复合体，不研究由多个瞬变引起的紧急行为 互动。为了应对这一挑战，我们使用DNA纳米技术支架来设计大分子图案 体外和一种新的遗传编码的ER/K连接子来探测和调节活细胞中的蛋白质相互作用。 总之，我们利用这些技术来剖析G蛋白多样性的分子机制- 细胞中非常规肌球蛋白功能的偶联受体(GPCR)信号和生物物理调节。 我们使用生物传感器成功地研究了细胞中gpr信号特异性的两个不同方面。 通过将GPCR和G蛋白元件与ER/K连接子连接而设计。首先，我们假设配体 稳定选择性地与一个或多个GαC末端对接的GPCR亚状态，以进行调整 下游通路的配基效力和效力。我们的目标是使用gpcr生物传感器的组合。 和多尺度分子动力学模拟来定义热点残基、结构基序和变构 GPCR和G蛋白中驱动信号特异性的通路。第二，我们的研究取得了进展 GPCR和效应器之间的并发和顺序相互作用在信号特异性上的作用。我们的目标 是结合gpcr生物传感器和传统药理学方法来剖析 G蛋白对GPCR信号转导的影响。总而言之，我们的研究提供了对GPCR信号特异性的见解 可用于基于结构的药物发现工作，以确定具有功能选择性的GPCR配体。 非传统肌球蛋白在许多细胞过程中都是必不可少的，包括膜运输、收缩、 和细胞分裂。肌球蛋白在细胞中的运动功能的定义受到两者的无数几何结构的挑战 肌动蛋白网络和货物，配以各种发动机-货物接口。我们使用模拟货物的DNA 纳米技术支架结合计算建模成功解剖机械 肌球蛋白合奏中的协调。我们将使用这些方法来深入了解生物物理调节 通过马达-货物接口的肌球蛋白功能。我们假设货物界面起分子作用。 调整肌球蛋白功能以匹配单个细胞过程的功能要求的模块。我们的 目标是通过与不同的货物适配器、Rab GTP酶和cell-GTP的相互作用来剖析肌球蛋白的调节。 衍生货物复合体。我们的研究将促进我们对细胞内紧急肌球蛋白功能的理解， 同时为细胞骨架马达的货物调节提供了广泛的理论框架。

PROJECT SUMMARY Cellular processes such as signaling and membrane traffic emerge from an ensemble of dynamic, transient protein-protein interactions in crowded cellular environments. Aberrant protein-protein interactions are frequently implicated in debilitating or fatal diseases such as diabetes, neurodegenerative diseases, and cancer. Established structural and cell biological techniques are mostly limited to dissecting the function of stable protein complexes, and do not investigate emergent behavior stemming from multiple transient interactions. To address this challenge, we use DNA nanotechnology scaffolds to pattern macromolecules in vitro and a novel genetically encoded ER/K linker to probe and modulate protein interactions in live cells. Together, we leverage these technologies to dissect the molecular mechanisms of multiplicity in G protein- coupled receptor (GPCR) signaling and biophysical regulation of unconventional myosin function in cells. We have successfully investigated two distinct aspects of GPCR signaling specificity in cells using biosensors engineered by linking GPCR and G protein elements with an ER/K linker. First, we hypothesize that ligands stabilize GPCR conformational sub-states that selectively interface with one or more Gα C-termini, to tune ligand efficacy and potency for downstream pathways. Our goal is to use a combination of GPCR biosensors and multi-scale molecular dynamics simulations to define hot-spot residues, structural motifs, and allosteric pathways in both the GPCR and G protein that drive signaling specificity. Second, our research has advanced a role for concurrent and sequential interactions between GPCR and effectors on signaling specificity. Our goal is to combine GPCR biosensors and traditional pharmacology approaches to dissect the synergistic effects of G proteins on GPCR signaling. Together, our research provides insights into GPCR signaling specificity that can be leveraged in structure-based drug discovery efforts to identify functionally selective GPCR ligands. Unconventional myosins are essential in numerous cellular processes including membrane traffic, contractility, and cell division. Defining the motile function of myosins in cells is challenged by the myriad geometries of both actin networks and cargo, paired with a diversity of motor-cargo interfaces. We use cargo-mimetic DNA nanotechnology scaffolds combined with computational modeling to successfully dissect the mechanical coordination in myosin ensembles. We will use these approaches to gain insight into the biophysical regulation of myosin function through the motor-cargo interface. We hypothesize that cargo interfaces act as molecular modules that tune myosin function to match the functional requirements of individual cellular processes. Our goal is to dissect myosin regulation through interactions with distinct cargo adaptors, Rab GTPases, and cell- derived cargo complexes. Our research will advance our understanding of emergent myosin function in cells, while providing a broad theoretical framework for the cargo-mediated regulation of cytoskeletal motors.