A key event in transducing changes in oxygen concentration to changes in red cell mass is Prolyl Hydroxylase Domain protein 2 (PHD2)-catalyzed hydroxylation of the transcription factor, Hypoxia Inducible Factor-2? (HIF-2?). When oxygen is plentiful, constitutive hydroxylation of HIF-2? by PHD2 targets the former for degradation. When oxygen is lacking, PHD2 activity is attenuated, leading to the stabilization of HIF-2? and the activation of the ERYTHROPOIETIN (EPO) gene in the kidney. Circulating EPO then binds to the EPO receptor on red cell progenitors to expand red cell mass. Our understanding of PHD2 is still at a very early stage. PHD2 contains two evolutionarily conserved domains, a prolyl hydroxylase domain that catalyzes the hydroxylation reaction, and a predicted zinc finger domain. Very little is known about the latter, which, it may be noted, is lacking in the other two mammalian PHD paralogues. We have recently found that the zinc finger domain, which is of the Myeloid Nervy Deaf (MYND) type, binds with high stringency to a Pro-Xaa-Leu-Glu motif that is present in select cochaperones of the HSP90 pathway, such as p23. The HIF-?'s (of which HIF-1? and HIF-2? are the major isoforms) are client proteins of the HSP90 pathway, and this therefore leads to a model in which p23 recruits PHD2 to HSP90 to promote HIF-? hydroxylation. Thus, the zinc finger of PHD2 is proposed to serve a positive regulatory role. Key unanswered questions are whether the zinc finger of PHD2 is actually of the MYND-type, and whether its function is relevant to erythropoiesis and more generally, oxygen homeostasis. We propose to address these questions by pursuing the following Specific Aims.
In Specific Aim 1, we will conduct point mutagenesis studies on the zinc finger of PHD2 to determine if residues that might be predicted to be functional important based on homology to known MYND zinc fingers are indeed important for interaction with the Pro-Xaa-Leu-Glu motif of p23.
In Specific Aim 2, we will characterize a knockin mouse line in which two predicted zinc chelating residues have been mutated (C36S/C42S) in order to ablate zinc finger function. We will examine this mouse for dysregulation of erythropoiesis, and cross it with Hif-1? and Hif-2? heterozygous deficient mice to determine Hif-? dependency.
In Specific Aim 3, we will determine whether erythroid progenitors obtained from these mice display hypersensitivity to Epo, which would point to an Epo-independent role for this zinc finger that is distinct from the regulation the Epo gene itself. We will also examine whether there is a broader upregulation of Hif target genes in tissues of these mice. Collectively, we anticipate that these studies will provide critical information for PHD2, a key signaling protein that regulates red cell mass.
The key oxygen sensor in the pathway that controls red blood cell mass is a protein called PHD2. Understanding the regulation of PHD2 will provide the basis for manipulating this pathway for therapeutic benefit in diseases such as anemia. We have recently identified a novel way by which PHD2 is regulated, and propose defining this new model of oxygen-sensing.
| Sinnema, Margje; Song, Daisheng; Guan, Wei et al. (2018) Loss-of-function zinc finger mutation in the EGLN1 gene associated with erythrocytosis. Blood 132:1455-1458 |
| Arsenault, Patrick R; Song, Daisheng; Bergkamp, Marian et al. (2016) Identification of Small-Molecule PHD2 Zinc Finger Inhibitors that Activate Hypoxia Inducible Factor. Chembiochem 17:2316-2323 |
| Arsenault, Patrick R; Song, Daisheng; Chung, Yu Jin et al. (2016) The Zinc Finger of Prolyl Hydroxylase Domain Protein 2 Is Essential for Efficient Hydroxylation of Hypoxia-Inducible Factor ?. Mol Cell Biol 36:2328-43 |
