Animal models to examine role of ZPR1 protein complexes

描述（由申请人提供）：拟议研究的长期目标是了解锌指蛋白ZPR1复合物在细胞生长和发育中的生理作用。ZPR1与存活运动神经元（SMN）和真核翻译延伸因子1A （eEF1A）蛋白相互作用。蛋白质之间的相互作用是哺乳动物正常生长发育所必需的生物学功能。突变导致蛋白质-蛋白质复合物的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是由存活运动神经元（SMN1）基因突变引起的，以脊髓运动神经元变性为特征。SMN突变导致SMA患者的SMN核积聚缺陷。ZPR1是SMN在细胞核中积累所必需的。在来自SMN突变的SMA患者的细胞中，ZPR1与SMN的相互作用被破坏。显然，ZPR1-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，ZPR1-SMN复合物的功能尚不清楚。ZPR1与eEF1A的相互作用是正常细胞生长和增殖所必需的。破坏ZPR1与eEF1A的相互作用会导致细胞生长缺陷，导致细胞在细胞周期的G2/M期积聚。ZPR1与eEF1A和eEF1A2（脑特异性异构体）相互作用。由于eEF1A2基因在消瘦（wst）小鼠中发生突变，eEF1A2表达丧失，导致进行性运动神经元变性和肌肉萎缩。ZPR1蛋白复合物在哺乳动物细胞生长发育中的确切作用尚不清楚。在这一提议中，我们将开发新的工具来检测ZPR1复合物与eEF1A和SMN蛋白在小鼠细胞生长和发育中的功能。我们将建立Zpr1敲入小鼠模型，选择性地破坏Zpr1蛋白复合物。在第一个特定目标中，我们将产生具有破坏Zpr1 - eef1a复合物突变的Zpr1敲入小鼠。我们利用ZPR1的x射线晶体结构在nh2末端区域确定了ZPR1与eEF1A相互作用所需的关键氨基酸残基。我们将创建具有双点突变和四点突变的Zpr1敲入小鼠，破坏Zpr1与eEF1A的相互作用，分别导致中度和重度细胞生长缺陷。在第二个具体目标中，我们将在ZPR1的cooh末端区域识别破坏ZPR1与SMN相互作用的点突变，设计和构建靶向载体，用于产生具有破坏ZPR1-SMN复合物突变的ZPR1敲入小鼠。Zpr1敲入小鼠模型的建立将允许在体内检测Zpr1蛋白复合物的生理功能，并确定Zpr1 - SMN和Zpr1 - eef1a复合物是否为哺乳动物的生存、生长和发育所必需。了解蛋白质复合物的细胞机制将促进生物医学研究领域的知识，包括作为治疗靶点的蛋白质-蛋白质相互作用。

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.