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: 8646929
• 负责人: JAMES A MCNEW
• 金额: $ 28.63万
• 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/8646929
• 关键词: Accounting; Amphipathic Alpha Helix; Area; Arginine; Automobile Driving; Back; Biochemical; Biogenesis; Biological Assay; Biophysical Process; 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（由申请人提供）：膜融合对于真核细胞生物学的许多方面都至关重要，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。膜融合还提供细胞质细胞器如线粒体和内质网改变形状和大小以执行其所需功能的能力。本研究探讨了GT3-atlastin在同型ER融合中的作用。SPG3A是一个更大的基因家族的成员，该基因家族负责一组称为遗传性痉挛性截瘫（HSP）的遗传性神经系统疾病。atlastin-1的突变占HSP常染色体显性形式的约10%。对atlastin在通过同型ER融合产生和维持ER功能中的作用的基本理解将显著地告知ER相关病理学的机制基础，例如在HSP中发现的神经元变性。 双肽蛋白利用GTP水解的化学能对磷脂双层做功。这种膜融合的新机制不同于任何已知的融合蛋白，并建立了一个新的范式。最近的结构工作使我们能够开发一个详细的模型atlastin功能。我们将使用重组蛋白、体外融合反应、GTdR活性测量和寡聚状态测定来测试该模型的重要预测。我们将探测特定的蛋白质的要求，通过保守的GTdR结构域，稳定的膜附着提供了一个三螺旋束段，连接GTdR结构域跨膜锚，膜不稳定的两亲性螺旋在C-末端胞质尾。 这项工作将有助于两个非常重要的领域， 研究，遗传性痉挛性截瘫的病理生理学和膜融合的一般机制。对HSP最主要形式的分子遗传学分析，包括atlastin，确定了通常参与ER功能的蛋白质。适当运作 ER对于所有细胞都是至关重要的，因为这个细胞器提供了关于分泌装置、脂质生物合成和钙稳态的关键活动。最近的数据表明，维持网状形态是ER功能所必需的，而atlastin部分地提供了改变形状和维持管腔连续性的能力。这种新的方式合并膜的特性将是重要的理解ER同型融合和ER稳态的生物物理机制一般。