Identifying Key Structural Elements in the Activation of the Arp2/3 Complex

识别 Arp2/3 复合体激活中的关键结构元件

• 批准号: 8218060
• 负责人: PATRICIA A KHUU
• 金额: $ 4.92万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8218060
• 关键词: ATP-G-actin; Actins; Address; Affect; Affinity; Amino Acids; Binding; Biochemical; Biological; Biological Assay; Bundling; Cell Shape; Cell physiology; Cells; Cellular Structures; Cicatrix; Complement; Complex; Confocal Microscopy; Crystallography; Cytoskeleton; DNA Sequence; Data; Daughter; Detection; Disease; Elements; Endocytosis; Exhibits; Face; Filament; Fission Yeast; Functional disorder; Gel Chromatography; Genes; Genotype; Goals; Hereditary Disease; Human; Immune system; Indium; Ion Exchange; Kinetics; Knock-out; Link; Measures; Microfilaments; Microscopy; Modeling; Molecular; Mothers; Mutation; Neoplasm Metastasis; Participant; Pathologic Processes; Peptides; Phenotype; Physiological Processes; Process; Protein Binding; Protein Family; Proteins; Research; Side; Site; Site-Directed Mutagenesis; Structure; Techniques; Western Blotting; Wiskott-Aldrich Syndrome; Yeasts; base; cancer cell; crosslink; depolymerization; gene replacement; homologous recombination; in vivo; insight; monomer; mutant; pathogen; polymerization; public health relevance; research study

DESCRIPTION (provided by applicant): As a nucleator of branched actin filaments, the actin-related protein2/3 (Arp2/3) complex fulfills in a key role in remodeling the actin filament network, a major component of the cellular cytoskeleton. The complex is essential for changes to cell shape associated with specific processes, such as endocytosis in yeast, and is required for the formation of actin-based structures, such as actin patches and lamellipodia. Our proposed research seeks to address the fundamental questions about the mechanistic basis of Arp2/3 activation that remain unanswered due to a lack of crucial biochemical and structural data of the active complex. Our goal is to determine the precise atomic interactions that activate the Arp2/3 complex. Accordingly, using Schizosaccharomyces pombe, the fission yeast, we will employ directed mutations of specific amino acid residues in the Arp2/3 complex to determine how mutations at these sites affect the binding of the complex to the sides of pre-existing (mother) actin filaments and nascent (daughter) actin filaments. We will use mutagenic primers for site-directed mutagenesis, along with homologous recombination, gene knock-out and gene replacement, to create mutant strains. PCR and DNA sequencing will be used to genotype mutant strains, while Western blot will reveal expression levels of mutant genes. Mutant complexes will be purified through GST-VCA affinity, ion exchange, and gel filtration chromatography. Phenotype analysis of mutants will be visualized with GFP-tagged markers using confocal microscopy and total internal reflection microscopy (TIRF). We will carry out spectroscopic assays to measure the kinetics of actin polymerization dynamics of mutant Arp2/3 complexes as well as their binding affinity to ATP, actin monomer, the pointed-end of the daughter filament and nucleating-promoting factors (NPFs), such as Wiskott- Aldrich syndrome protein (WASP) family. Additionally, we will use x-ray crystallography to determine the structural basis for Arp2/3 activation by WASP family proteins for some mutants identified in the mother and daughter filament binding experiments that exhibit high affinity for VCA, an activating region of the WASP proteins. The results of our study will offer a detailed model of the interfaces between the Arp2/3 complex and the mother and daughter actin filaments and how WASP proteins bind to the complex to contribute valuable insight to understanding how the Arp2/3 complex is regulated. PUBLIC HEALTH RELEVANCE: The Arp2/3 complex is widely conserved and essential for eukaryotic cellular function; consequently, its dysfunction is likely to be involved in the physiological and pathological processes of many diseases. A number of human pathogens usurp the power of Arp2/3 complex to infect cells or evade detection by the immune system [1-3], while mutations in WASp, an activator of Arp2/3 complex, result in the Wiskott-Aldrich Syndrome (WAS), an X-linked genetic disorder[4]. Arp2/3 is also involved in metastasis as it is required in the formation of cellular structures such as lamellipodia of migrating cells and invadopodia of cancers cells [5, 6].

描述（由申请人提供）：作为分支肌动蛋白丝的成核器，与肌动蛋白相关的蛋白2/3（ARP2/3）复合物在重塑肌动蛋白丝网络（细胞细胞膜的主要成分）方面具有关键作用。该复合物对于改变与特定过程相关的细胞形状（例如酵母中内吞作用）至关重要，并且是基于肌动蛋白的结构（例如肌动蛋白斑块和层状脂蛋白）形成所必需的。我们提出的研究旨在解决有关ARP2/3激活的机理基础的基本问题，由于缺乏活性复合物的重要生化和结构数据，该问题仍未得到解答。我们的目标是确定激活ARP2/3复合物的精确原子相互作用。 因此，使用裂变酵母的岩石酵母POMBE，我们将采用ARP2/3复合物中特定氨基酸残基的定向突变，以确定这些位点的突变如何影响该络合物与静态（母）肌动蛋白丝和Nascent（Nascent（Nascent（Nascent）（nascent）（女儿）肌动蛋白丝的结合。我们将使用诱变引物进行定位的诱变，以及同源重组，基因敲除和基因置换，以产生突变菌株。 PCR和DNA测序将用于基因型突变菌株，而Western印迹将揭示突变基因的表达水平。突变复合物将通过GST-VCA亲和力，离子交换和凝胶过滤色谱法纯化。使用共聚焦显微镜和总内反射显微镜（TIRF），将使用GFP标记的标记可视化突变体的表型分析。我们将进行光谱测定法，以测量突变体ARP2/3复合物的肌动蛋白聚合动力学的动力学以及它们与ATP的结合亲和力，肌动蛋白单体，dopation细丝的尖端和成核的末端和成核促进性启动因子（NPF），例如WikeT-Aldrich-aldrich protine protine protine protine protine protine normal frompalsect。此外，我们将使用X射线晶体学来确定WASP家族蛋白激活ARP2/3的结构基础，用于在母女丝结合实验中鉴定出的某些突变体，这些突变体对VCA表现出高亲和力，VCA是WASP蛋白的激活区域。我们的研究结果将提供ARP2/3复合物与母女肌动蛋白丝之间的界面的详细模型，以及WASP蛋白如何与复合物结合，从而为了解ARP2/3复合物的调节如何受到宝贵的见识。 公共卫生相关性：ARP2/3复合物广泛保守，对于真核细胞功能至关重要；因此，其功能障碍可能与许多疾病的生理和病理过程有关。许多人病原体篡夺了ARP2/3复合物在感染细胞或通过免疫系统逃避检测的功率[1-3]，而WASP中的突变是ARP2/3复合物的激活剂，导致Wiskott-Aldrich综合征（WAS），一种X-C-连接的遗传疾病[4]。 ARP2/3也参与转移，因为它在形成细胞结构（例如迁移细胞的薄片和癌细胞的侵袭性）等细胞结构中需要它[5，6]。