Animal models to examine role of ZPR1 protein complexes

检查 ZPR1 蛋白复合物作用的动物模型

• 批准号: 7640364
• 负责人: Laxman Dass Gangwani
• 金额: $ 18.38万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-08 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7640364
• 关键词: Address; Amino Acids; Animal Model; Biochemical; Biological Process; Biomedical Research; Brain; Cell Cycle Regulation; Cell Nucleus; Cell physiology; Cells; Cessation of life; Complex; DNA biosynthesis; Data; Defect; Development; Diabetes Mellitus; Disease; Drug Delivery Systems; Elongation Factor; Embryo; Exons; Generations; Genes; Genetic; Genetic Transcription; Goals; Growth and Development function; Heart Diseases; Hereditary Disease; Human Genetics; In Vitro; Knock-in Mouse; Knockout Mice; Knowledge; Malignant Neoplasms; Mammalian Cell; Mammals; Mitosis; Motor Neurons; Mus; Muscular Atrophy; Mutation; N-terminal; Nerve Degeneration; Neurodegenerative Disorders; Normal Cell; Nuclear; Patients; Physiological; Point Mutation; Protein Isoforms; Proteins; RNA Splicing; Research; Research Proposals; Roentgen Rays; Role; SMN protein (spinal muscular atrophy); SMN1 gene; Signal Transduction; Site-Directed Mutagenesis; Spinal; Spinal Muscular Atrophy; Structure; Tissues; Translations; Yeasts; Zinc Fingers; cell growth; design and construction; disease-causing mutation; genetic analysis; human disease; in vivo; insight; motor neuron degeneration; mouse model; novel; protein complex; protein protein interaction; public health relevance; therapeutic target; tool; vector; wasting

DESCRIPTION (provided by applicant): The long-term goal of proposed research is to understand the physiological role of zinc finger protein ZPR1 complexes in cell growth and development. ZPR1 interacts with survival motor neuron (SMN) and eukaryotic translation elongation factor 1A (eEF1A) proteins. Protein-protein interactions are essential for biological functions required for normal growth and development of mammals. Disruption of protein-protein complexes due to mutations is a major cause of diverse human genetic diseases ranging from cancer to neurodegenerative disorders, including spinal muscular atrophy (SMA). SMA is caused by mutations of the survival motor neurons (SMN1) gene and characterized by degeneration of spinal motor neurons. Mutations in SMN cause defects in nuclear accumulation of SMN in patients with SMA. ZPR1 is required for accumulation of SMN in the nucleus. Interaction of ZPR1 with SMN is disrupted in cells derived from SMA patients that have SMN mutations. It is clear that the formation of ZPR1-SMN complexes is critical for nuclear accumulation and normal function of SMN. However, the function of ZPR1-SMN complexes is unknown. Interaction of ZPR1 with eEF1A is required for normal cell growth and proliferation. Disruption of interaction between ZPR1 and eEF1A causes defects in cell growth and result in accumulation of cells in G2/M phase of the cell cycle. ZPR1 interacts with both eEF1A and eEF1A2 (brain specific isoform). Loss of eEF1A2 expression, due to mutation of the eEF1A2 gene in wasted (wst) mice, results in progressive motor neuron degeneration and muscle atrophy. The precise role of ZPR1 protein complexes in mammalian cell growth and development is unclear. In this proposal, we will develop new tools to examine the function of ZPR1 complexes with eEF1A and SMN proteins in cell growth and development using genetic analysis in mice. We will generate Zpr1 knock-in mouse models to selectively disrupt ZPR1 protein complexes. In the first specific aim, we will generate Zpr1 knock-in mice with mutations that disrupt ZPR1-eEF1A complexes. We have identified critical ZPR1 amino acid residues in the NH2-terminal region using the X-ray crystal structure of ZPR1 that are required for interaction of ZPR1 with eEF1A. We will create Zpr1 knock-in mice with a double point mutation and a quadruple point mutation that disrupts interaction of ZPR1 with eEF1A and result in moderate and severe defects in cell growth, respectively. In the second specific aim, we will identify point mutations in the COOH-terminal region of ZPR1 that disrupt interaction of ZPR1 with SMN to design and construct targeting vector for generation of Zpr1 knock-in mouse with mutations that disrupt ZPR1-SMN complexes. The development of Zpr1 knock-in mouse models would allow in vivo examination of physiological functions of ZPR1 protein complexes and determine whether ZPR1- SMN and ZPR1-eEF1A complexes are required for survival, growth and development in mammals. Understanding the cellular mechanisms of protein complexes will advance knowledge in the field of biomedical research, including protein-protein interaction as therapeutic target. PUBLIC HEALTH RELEVANCE: Many human genetic diseases are caused by mutations that result in alteration of protein-protein interactions, including spinal muscular atrophy (SMA). Mutations in SMN protein cause disruption of SMN complexes, including SMN-ZPR1 complexes in patients with SMA. Interaction of zinc finger protein ZPR1 with SMN is required for nuclear accumulation and normal function of SMN. Interaction of ZPR1 with eEF1A is required for normal cell growth. In this proposal, we will develop novel knock-in mouse models to examine functions of SMN-ZPR1 and ZPR1-eEF1A protein complexes. Understanding cellular function of protein complexes would allow use of protein-protein interactions as drug targets.

描述（由申请人提供）：拟议研究的长期目标是了解锌指蛋白ZPR 1复合物在细胞生长和发育中的生理作用。ZPR 1与运动神经元存活蛋白（SMN）和真核翻译延伸因子1A（eEF 1A）蛋白相互作用。蛋白质-蛋白质相互作用对于哺乳动物正常生长和发育所需的生物学功能是必不可少的。突变导致的蛋白质-蛋白质复合物破坏是多种人类遗传疾病的主要原因，从癌症到神经退行性疾病，包括脊髓性肌萎缩症（SMA）。SMA是由运动神经元存活基因（SMN 1）突变引起的，其特征是脊髓运动神经元变性。SMN突变导致SMA患者SMN核积聚缺陷。ZPR 1是SMN在细胞核中积累所必需的。ZPR 1与SMN的相互作用在源自具有SMN突变的SMA患者的细胞中被破坏。很明显，ZPR 1-SMN复合物的形成对于核积累和SMN的正常功能至关重要。然而，ZPR 1-SMN复合物的功能尚不清楚。ZPR 1与eEF 1A的相互作用是正常细胞生长和增殖所必需的。ZPR 1和eEF 1A之间相互作用的破坏导致细胞生长缺陷，并导致细胞在细胞周期的G2/M期积累。ZPR 1与eEF 1A和eEF 1A 2（脑特异性亚型）相互作用。由于消瘦（wst）小鼠中eEF 1A 2基因突变导致eEF 1A 2表达缺失，导致进行性运动神经元变性和肌肉萎缩。ZPR 1蛋白复合物在哺乳动物细胞生长和发育中的确切作用尚不清楚。在这项提案中，我们将开发新的工具，利用小鼠的遗传分析来研究ZPR 1与eEF 1A和SMN蛋白复合物在细胞生长和发育中的功能。我们将产生ZPR 1基因敲入小鼠模型，以选择性地破坏ZPR 1蛋白复合物。在第一个具体目标中，我们将产生具有破坏ZPR 1-eEF 1A复合物的突变的Zpr 1敲入小鼠。我们已经确定了关键的ZPR 1氨基酸残基的NH 2-末端区域使用的X-射线晶体结构的ZPR 1所需的ZPR 1与eEF 1A的相互作用。我们将创建具有双点突变和四点突变的Zpr 1敲入小鼠，其破坏ZPR 1与eEF 1A的相互作用，并分别导致细胞生长的中度和重度缺陷。在第二个具体目标中，我们将鉴定ZPR 1的COOH末端区域中破坏ZPR 1与SMN相互作用的点突变，以设计和构建靶向载体，用于产生具有破坏ZPR 1-SMN复合物的突变的ZPR 1敲入小鼠。ZPR 1基因敲入小鼠模型的开发将允许在体内检查ZPR 1蛋白复合物的生理功能，并确定ZPR 1- SMN和ZPR 1-eEF 1A复合物是否是哺乳动物生存、生长和发育所必需的。了解蛋白质复合物的细胞机制将推进生物医学研究领域的知识，包括蛋白质-蛋白质相互作用作为治疗靶点。 公共卫生关系：许多人类遗传性疾病是由导致蛋白质-蛋白质相互作用改变的突变引起的，包括脊髓性肌萎缩症（SMA）。SMN蛋白突变导致SMN复合物破坏，包括SMA患者的SMN-ZPR 1复合物。锌指蛋白ZPR 1与SMN的相互作用是核积累和SMN正常功能所必需的。ZPR 1与eEF 1A的相互作用是正常细胞生长所必需的。在这项提议中，我们将开发新的基因敲入小鼠模型来研究SMN-ZPR 1和ZPR 1-eEF 1A蛋白复合物的功能。了解蛋白质复合物的细胞功能将允许使用蛋白质-蛋白质相互作用作为药物靶点。