Biochemical analysis of the ER fusion protein allastin

ER 融合蛋白阿拉斯丁的生化分析

• 批准号: 8460000
• 负责人: JAMES A MCNEW
• 金额: $ 27.65万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8460000
• 关键词: Accounting; Amphipathic Alpha Helix; Area; Arginine; Automobile Driving; Back; Biochemical; Biogenesis; Biological Assay; C-terminal; Calcium; Catalysis; Cells; Cellular biology; Chemicals; Chimeric Proteins; Crosslinker; Cryoelectron Microscopy; Cytoplasmic Organelle; Cytoplasmic Tail; Data; Dependence; Disease; Docking; Drosophila genus; Endoplasmic Reticulum; Endosomes; Eukaryotic Cell; Functional disorder; Gene Family; Generations; Genetic; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hereditary Spastic Paraplegia; Homeostasis; Hydrolysis; In Vitro; Inherited; Length; Lipids; Lower Extremity; Lysosomes; Maintenance; Measurement; Mediating; Membrane; Membrane Fusion; Mitochondria; Modeling; Molecular Analysis; Molecular Genetics; Monitor; Morphology; Mutation; Neck; Nerve Degeneration; Nucleotides; Organelles; Pathology; Pathway interactions; Phospholipids; Play; Proteins; Reaction; Recombinant Proteins; Recombinants; Research; Role; SNAP receptor; Shapes; Staging; System; Tail; Testing; Transmembrane Domain; Viral Fusion Proteins; Work; base; design; dimer; genetic analysis; inhibitor/antagonist; insight; interfacial; member; mutant; nervous system disorder; novel; prevent; protein folding; trafficking

DESCRIPTION (provided by applicant): Membrane fusion is vitally important for many aspects of eukaryotic cell biology including vesicular traffic within the secretory pathway as well as the biogenesis and maintenance of the entire endomembrane system. Membrane fusion also provides cytoplasmic organelles like mitochondria and the endoplasmic reticulum the ability to change shape and size to perform their required function. This proposal examines the role of the GTPase atlastin in homotypic ER fusion. Atlastin (SPG3A) is a member of a larger family of genes that are responsible for a group of inherited neurological disorders called Hereditary Spastic Paraplegias (HSP). Mutations in atlastin-1 account for ~10% of autosomal dominant forms of HSP. A fundamental understanding of atlastin's role in generating and maintaining ER function by homotypic ER fusion will significantly inform the mechanistic basis of ER-associated pathologies such as the neuronal degeneration found in HSP. Atlastin utilizes the chemical energy of GTP hydrolysis to do work on the phospholipid bilayer. This novel mechanism of membrane fusion is unlike any known fusion protein and establishes a new paradigm. Recent structural work has allowed us to develop a detailed model of atlastin function. We will test important predictions of this model using recombinant proteins, in vitro fusion reactions, measurement of GTPase activity, and determination of oligomeric state. We will probe the specific protein requirements for membrane tethering through the conserved GTPase domain, stable membrane attachment provided by a three helical bundle segment that connects the GTPase domain to transmembrane anchors, and membrane destabilization by an amphipathic helix in the C-terminal cytoplasmic tail. This work will contribute to two very important areas of research, the pathophysiology of Hereditary Spastic Paraplegia and the general mechanism of membrane fusion. Molecular genetic analysis of the most prominent forms of HSP, including atlastin, identified proteins that are generally involved in ER function. Proper functioning of the ER is critical for all cells given the crucial activities this organelle provides with respect to te secretory apparatus, lipid biogenesis, and calcium homeostasis. Recent data suggest that maintenance of a reticular morphology is necessary for ER function and atlastin, in part, provides for the ability to change shape and maintain lumen continuity. Characterization of this new way to merge membranes will be important for understanding the biophysical mechanisms of ER homotypic fusion and ER homeostasis in general.

描述(申请人提供)：膜融合对于真核细胞生物学的许多方面都是至关重要的，包括分泌途径中的囊泡运输。 作为整个内膜系统的生物发生和维持。膜融合还为线粒体和内质网等细胞质细胞器提供了改变形状和大小以执行其所需功能的能力。这项建议研究了GTP酶atlastin在同型ER融合中的作用。Atlastin(SPG3A)是一个更大的基因家族的成员，该家族负责一组遗传性神经疾病，称为遗传性痉挛麻痹(HSP)。Atlastin-1基因突变约占常染色体显性遗传型HSP的10%。对ATLASTIN在同型ER融合产生和维持ER功能中的作用的基本了解将显著地为ER相关病理的机制基础提供信息，例如在HSP中发现的神经元变性。Atlastin利用GTP水解的化学能在磷脂双层上做功。这种膜融合的新机制不同于任何已知的融合蛋白，并建立了一种新的范式。最近的结构工作使我们能够开发出atlastin功能的详细模型。我们将使用重组蛋白、体外融合反应、GTP酶活性的测量和寡聚体状态的确定来测试这个模型的重要预测。我们将通过保守的GTPase结构域探索膜系留的特定蛋白质需求，通过将GTPase结构域连接到跨膜锚的三螺旋束片段提供稳定的膜附着，以及通过C末端细胞质尾部的两亲性螺旋提供膜的不稳定性。这项工作将有助于以下两个非常重要的领域 遗传性痉挛截瘫的病理生理学研究和膜融合的一般机制。对包括阿特拉斯汀在内的最突出形式的热休克蛋白进行分子遗传学分析，确定了通常与内质网功能有关的蛋白质。的正常运作 内质网对所有细胞都是至关重要的，因为这个细胞器提供了与TE分泌器、脂质生物发生和钙稳态有关的关键活动。最近的数据表明，维持网状形态对于ER的功能是必要的，而atlastin在一定程度上提供了改变形状和维持管腔连续性的能力。这种新的膜合并方式的表征对于理解内质网同型融合和内质网内稳态的生物物理机制具有重要意义。