Arf Functional Landscapes

Arf功能景观

• 批准号: 10408843
• 负责人: CATHERINE A ROYER
• 金额: $ 34.95万
• 依托单位: RENSSELAER POLYTECHNIC INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10408843
• 关键词: ADP-Ribosylation Factors; Alzheimer' s Disease; Autoimmune Diseases; Bacteria; Binding; Biophysics; Brefeldin A; Cell Shape; Cell membrane; Cell physiology; Cells; Cerebral cortex; Chemicals; Communicable Diseases; Cytokinesis; Data; Development; Disease; Endosomes; Enzymes; Eukaryotic Cell; Exhibits; Fluorescence; Free Energy; GTP Binding; GTPase-Activating Proteins; Genetic; Goals; Golgi Apparatus; Grain; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hepatitis C virus; Human; Infection; Invaded; Investigation; Link; Lipids; Malignant Neoplasms; Maps; Membrane; Mental Retardation; Mitochondria; Modeling; Modification; Molecular; Molecular Conformation; Monomeric GTP-Binding Proteins; Multidimensional NMR Techniques; Mutation; Mutation Analysis; N-terminal; Nucleotides; Organelles; Parkinson Disease; Pathway interactions; Pharmaceutical Preparations; Population; Positioning Attribute; Protein Conformation; Proteins; Regulation of Cell Size; Salmonella; Signal Pathway; Signal Transduction; Site; Solvents; Specificity; Structure; System; Therapeutic; Virus; Yeasts; base; biophysical tools; lipid transport; malformation; molecular dynamics; molecular recognition; nervous system disorder; novel; pathogenic bacteria; pathogenic virus; pressure; protein structure; three dimensional structure; tool; trafficking; vesicle transport

Summary Membrane organization in eukaryotic cells is controlled by ADP ribosylation factors (Arfs), small GTPases that function as molecular switches to activate signaling cascades. Arfs regulate vesicular transport of lipids and proteins between the ER and the Golgi (Class I-Arf1) and endosome-plasma membrane trafficking (Class III-Arf6), implicating Arf function in cytokinesis, cell shape, organelle transport, mitochondrial and lipid droplet function and pH-dependent regulation of cell size. Mutations in Arfs or their partners have been linked to genetic neurological diseases causing severe malformation of the cerebral cortex or mental retardation. Moreover, many pathogenic bacteria and viruses commandeer Arfs as they invade cells, thereby promoting infection. Our overall goal is to understand the nucleotide exchange transitions of Arf GTPases, the mechanisms of which cannot be deduced from their static structures. We hypothesize that the Arf conformations specifically recognized by their cognate exchange factors correspond to significantly disrupted excited states that are populated at very low levels under standard conditions. Specifically, we aim to map the GDP/GTP switches of Arf1 and Arf6 (Aims 1 and 2), and using mutational analysis, establish the underlying molecular mechanisms of their functional specificity (Aim 3). Our approach combines experimental biophysical tools (multi- dimensional NMR, SAXS and fluorescence) with pressure perturbation and coarse-grained molecular dynamics simulations constrained by our data, to provide structural ensembles and pseudo-free energy landscapes that will reveal functionally relevant excited states implicated in Arf function and specificity. These excited state structures will provide novel target sites for inhibiting Arf signaling pathways, offering new avenues for developing approaches to mitigate the invasive capacity of bacteria and viruses. More generally, the pressure-based mapping approach proposed here represents a powerful means to characterize elusive states of proteins implicated in their functions.

摘要 真核细胞的膜组织由ADP核糖化因子(ARF)控制，小的 GTP酶起分子开关的作用，激活信号级联。ARF调节水泡 内质网与高尔基体(I-Arf1类)和内体-血浆之间的脂类和蛋白质转运 膜转运(III-Arf6类)，涉及Arf在胞质分裂、细胞形状、细胞器中的功能 运输、线粒体和脂滴功能以及对细胞大小的pH依赖性调节。突变 ARF或其伴侣与导致严重畸形的遗传性神经疾病有关 大脑皮层或精神发育迟滞。此外，许多致病细菌和病毒 当ARF侵入细胞时，它们会被征用，从而促进感染。我们的总体目标是了解 Arf GTP酶的核苷酸交换转换，其机制尚不能从 它们的静态结构。我们假设Arf构象是由它们的 同源交换因子对应于在很大程度上被破坏的激发态 标准条件下的低水平。具体来说，我们的目标是映射Arf1和GTP的GDP/GTP交换机 ARF6(目标1和2)，并使用突变分析，建立了潜在的分子机制 它们的功能特异性(目标3)。我们的方法结合了实验生物物理工具(多个 二维核磁共振、小角X射线散射和荧光)与压力微扰和粗粒分子 受我们的数据约束的动力学模拟，以提供结构系综和伪自由能 将揭示与Arf功能和特异性有关的功能相关激发态的景观。 这些激发态结构将为抑制Arf信号通路提供新的靶点， 为开发减轻细菌入侵能力的方法提供了新的途径 病毒。更广泛地说，这里提出的基于压力的映射方法代表了一种强大的 描述与其功能有关的蛋白质难以捉摸的状态的方法。