Animal models to examine role of ZPR1 protein complexes

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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.