In this proposal we present evidence, which supports a new model for the role of BCL2-related proteins in erythroid maturation. We show that both pro- and anti-apoptotic BCL2-related proteins are upregulated during terminal differentiation. One of these is the pro-apoptotic BH3-only protein, NIX. To see its role in development, we generated mice with targeted disruption of the Nix gene. These mice are viable, but have mild anemia and striking reticulocytosis. In the first aim, we propose experiments to characterize the defect in erythroid maturation in these mice. We propose to examine erythrocyte survival, to suppress erythropoiesis through hypertransfusion, and to stress the mice with phlebotomy and phenylhydrazine. We also propose to examine the maturation defect at the cellular level by isolating nascent reticulocytes, then examining their ultrastructure by electron microscopy as they mature. The model we propose is that the simultaneous increase in pro- and anti-apoptotic BCL2-related proteins induces autophagy, which is necessary for the remodeling of late erythroblasts. In the second aim, we propose several genetic experiments to test this hypothesis. We propose to breed Nix and Bcl-X mice to see if NIX is responsible for apoptosis in the absence of BCL-XL or under conditions of cytokine deprivation. We propose to breed Nix and Puma mice, to see if there is redundancy between these BH3-only proteins. Finally, we propose to breed Nix and conditional Bcl-X mice to if BCL-XL has a role in autophagy. Relevance: BCL-XL is essential for the development of erythroid cells. The prevailing model is that the primary function of BCL-XL is to provide a survival signal downstream of the erythropoietin receptor. There are studies, however, which suggest that BCL-XL functions late in erythroid differentiation, beyond the point where erythropoietin signaling is required. Now we show that another highly-regulated BCL2-related protein has role in late erythroid maturation. Together, these studies suggest that BCL2-related proteins, including BCL-XL, may have a fundamentally different role in erythroid differentiation. We propose to explore that possibility in experiments with Nix mice and other BCL2-related mouse strains. ? ? ?

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Exploratory/Developmental Grants (R21)
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Erythrocyte and Leukocyte Biology Study Section (ELB)
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Bishop, Terry Rogers
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St. Jude Children's Research Hospital
United States
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Ney, Paul A (2015) Mitochondrial autophagy: Origins, significance, and role of BNIP3 and NIX. Biochim Biophys Acta 1853:2775-83
Zhang, Ji; Loyd, Melanie R; Randall, Mindy S et al. (2012) A short linear motif in BNIP3L (NIX) mediates mitochondrial clearance in reticulocytes. Autophagy 8:1325-32
Zhang, Ji; Ney, Paul A (2011) Mechanisms and biology of B-cell leukemia/lymphoma 2/adenovirus E1B interacting protein 3 and Nip-like protein X. Antioxid Redox Signal 14:1959-69
Ney, Paul A (2011) Normal and disordered reticulocyte maturation. Curr Opin Hematol 18:152-7
Joo, Joung Hyuck; Dorsey, Frank C; Joshi, Aashish et al. (2011) Hsp90-Cdc37 chaperone complex regulates Ulk1- and Atg13-mediated mitophagy. Mol Cell 43:572-85
Zhang, Ji; Ney, Paul A (2010) Reticulocyte mitophagy: monitoring mitochondrial clearance in a mammalian model. Autophagy 6:405-8
Novak, Ivana; Kirkin, Vladimir; McEwan, David G et al. (2010) Nix is a selective autophagy receptor for mitochondrial clearance. EMBO Rep 11:45-51
Zhang, Ji; Randall, Mindy S; Loyd, Melanie R et al. (2009) Mitochondrial clearance is regulated by Atg7-dependent and -independent mechanisms during reticulocyte maturation. Blood 114:157-64
Zhang, J; Ney, P A (2009) Role of BNIP3 and NIX in cell death, autophagy, and mitophagy. Cell Death Differ 16:939-46
Zhang, Ji; Kundu, Mondira; Ney, Paul A (2009) Mitophagy in mammalian cells: the reticulocyte model. Methods Enzymol 452:227-45

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