BAR PROTEINS LINKING MEMBRANE AND CYTOSKELETON DYNAMICS

连接膜和细胞骨架动力学的条蛋白

• 批准号: 9174446
• 负责人: ROBERTO DOMINGUEZ
• 金额: $ 40.25万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-15 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9174446
• 关键词: AMPA Receptors; Actins; Alzheimer' s Disease; Architecture; Banana; Binding; Binding Proteins; Biochemical; Biochemistry; Brain; Brain Diseases; C-terminal; Cells; Cellular biology; Cytoskeleton; Dendritic Spines; Development; Dimerization; Drug Addiction; Epilepsy; Family; Fluorescence Resonance Energy Transfer; Goals; Grant; Guanosine Triphosphate Phosphohydrolases; Health; Heterodimerization; Human; In Vitro; Learning; Maps; Mediating; Membrane; Memory; Molecular; Morphogenesis; Myosin Type II; N-Methyl-D-Aspartate Receptors; N-terminal; Neurodegenerative Disorders; Neurons; Organelles; Phosphorylation; Phosphorylation Site; Play; Process; Proteins; Research; Role; SH3 Domains; Shapes; Side; Signal Transduction; Structure; Synapses; Synaptic Receptors; Synaptic Transmission; Tail; Tertiary Protein Structure; Testing; Work; base; biophysical techniques; brain tissue; cell motility; chronic pain; combinatorial; dimer; link protein; nanobodies; non-muscle myosin; protein protein interaction; receptor; response; rho; sensor; structural biology; trafficking

Project Summary Our goal is to determine the structural-functional mechanism by which BAR domain proteins coordinate actin cytoskeleton and membrane dynamics under the control of signaling cascades. The focus will be on two BAR domain proteins, PICK1 and IRSp53, involved in synaptic receptor trafficking and dendritic spine morphogenesis. Recent findings show that PICK1 is regulated by interaction with another BAR domain protein, ICA69, whereas IRSp53 is regulated by phosphorylation-dependent interaction with 14-3-3. Accordingly, we will investigate these interactions and how they modulate the activities of PICK1 and IRSp53. Our approach will span biochemistry, structural and cell biology, allowing us to correlate structure to function. PICK1 contains an N-terminal PDZ domain, a classical banana-shaped BAR domain, and an acidic C- terminal tail (ACT). IRSp53 contains an inverted BAR domain, followed by CRIB-PR and SH3 domains. PICK1 regulates the trafficking of several neuronal proteins, including AMPAR, thus playing a key role in controlling synaptic strength. IRSp53 regulates the formation of dendritic spines, and is an important modulator of NMDAR-mediated synaptic transmission, learning and memory. Extensive preliminary work underpins our hypotheses and supports feasibility. Our Aims are: Aim 1. Test the hypothesis that the ACT of PICK1 plays a dual role – inhibition of membrane binding and association (direct or indirect) with non-muscle myosin-II (NMII) for motility. We will determine the structure of PICK1 and the mechanism by which NMII drives the motility of PICK1-associated organelles. Anti-PICK1 nanobodies will be used in structural, biochemical and cellular studies. Aim 2. Determine the mechanism of interaction of PICK1 with ICA69/ICA1L. We hypothesize that PICK1 and ICA69/ICA1L interact as side-by-side homodimers, and not through heterodimerization as proposed. We will use several biophysical methods to test this hypothesis. Many BAR proteins associate in this manner, offering a general combinatorial mechanism for modulation of membrane curvature and partner recruitment. Aim 3. Test the hypothesis that phosphorylation-dependent binding of 14-3-3 inhibits the interactions of IRSp53 with cytoskeletal effectors and Cdc42. We have developed an IRSp53 FRET-sensor that together with cellular studies allow us to determine the effects of 14-3-3 binding on the structure of IRSp53 and its ability to interact with cytoskeletal effectors and Cdc42 in vitro and in cells. Aim 4. Test the hypothesis that the symmetric 14-3-3 dimer binds asymmetrically to IRSp53, interacting with two distinct phosphorylation sites. We will map the phosphorylation sites in cells and determine the structural-functional bases for the asymmetric interaction of 14-3-3 with phospho-IRSp53.

项目摘要 我们的目标是确定BAR结构域蛋白协调的结构-功能机制 肌动蛋白细胞骨架和信号级联控制下的膜动力学。重点将放在 两种BAR结构域蛋白PICK 1和IRSp 53参与突触受体运输和树突状细胞 棘形态发生 最近的发现表明PICK 1通过与另一种BAR结构域蛋白ICA 69相互作用来调节， 而IRSp 53通过与14-3-3的磷酸化依赖性相互作用来调节。因此，我们将 研究这些相互作用以及它们如何调节PICK 1和IRSp 53的活性。我们的方法 将跨越生物化学，结构和细胞生物学，使我们能够将结构与功能联系起来。 PICK 1含有一个N-末端PDZ结构域，一个经典的香蕉形BAR结构域，和一个酸性的C- 末端尾（ACT）。IRSp 53含有反向BAR结构域，随后是CRIB-PR和SH 3结构域。 PICK 1调节包括AMPAR在内的几种神经元蛋白的运输，因此在神经元的生长和发育中发挥关键作用。 控制突触强度IRSp 53调节树突棘的形成，并且是一种重要的免疫调节因子。 NMDAR介导的突触传递、学习和记忆的调节剂。广泛的前期工作 支持我们的假设和可行性。我们的目标是： 目标1.检验PICK 1的ACT发挥双重作用的假设-抑制膜结合， 与非肌肌球蛋白II（NMII）的运动性相关（直接或间接）。康贝特人将以 PICK 1的结构和NMII驱动PICK 1相关细胞器运动的机制。 抗PICK 1纳米抗体将用于结构、生物化学和细胞研究。 目标2.确定PICK 1与ICA 69/ICA 1 L相互作用的机制。我们假设PICK 1 和ICA 69/ICA 1 L作为并排同源二聚体相互作用，而不是通过所提出的异源二聚化。 我们将使用几种生物物理学方法来验证这一假设。许多BAR蛋白与此相关， 的方式，提供了一个通用的组合机制，用于调制膜曲率和合作伙伴 招聘 目标3.检验14-3-3的磷酸化依赖性结合抑制以下相互作用的假设： IRSp 53与细胞骨架效应子和Cdc 42。我们开发了一种IRSp 53 FRET传感器， 细胞研究使我们能够确定14-3-3结合对IRSp 53结构的影响， 在体外和细胞中与细胞骨架效应物和Cdc 42相互作用的能力。 目标4。检验对称14-3-3二聚体不对称结合IRSp 53，相互作用的假设 有两个不同的磷酸化位点我们将绘制细胞中的磷酸化位点，并确定 14-3-3与磷酸-IRSp 53的不对称相互作用的结构-功能基础。