Control of Erythropoiesis by the Oxygen Sensor PHD2

通过氧传感器 PHD2 控制红细胞生成

• 批准号: 10295385
• 负责人: FRANK S LEE
• 金额: $ 40.63万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10295385
• 关键词: Address; Alleles; Altitude; Amino Acid Substitution; Anemia; Binding; Blood; C-terminal; Catalytic Domain; Cell Count; Client; Clinical Treatment; Complex; Erythrocyte Indices; Erythrocytes; Erythropoiesis; Event; Foundations; Genes; Heat-Shock Proteins 90; Hematocrit procedure; Hemoglobin; Human; Hydroxylation; Hypoxia; Hypoxia Inducible Factor; Impairment; In Vitro; Individual; Knock-in; Knock-in Mouse; Left; Link; Messenger RNA; Missense Mutation; Modeling; Molecular Chaperones; Mus; Mutation; N-terminal; Output; Oxygen; Pathway interactions; Patients; Procollagen-Proline Dioxygenase; Protein Isoforms; Proteins; Red Blood Cell Count; Red Cell Mass result; Regulation; Renal Interstitial Cell; Reporting; Research; Ribosomes; Role; Serum; Source; Tacrolimus Binding Proteins; Testing; Time; Tissues; Translations; Ursidae Family; Variant; Work; Zinc Fingers; insight; interest; kidney cortex; loss of function mutation; polypeptide; prevent; protein folding; recruit; sensor; transcription factor

PROJECT SUMMARY/ABSTRACT The central oxygen sensor that controls red cell mass is PHD2 (also known as EGLN1). In specialized interstitial cells of the renal cortex, PHD2 prolyl hydroxylates the transcription factor HIF-2a in an oxygen-dependent manner and targets it for degradation. Under hypoxic conditions, prolyl hydroxylation is arrested, leading to the stabilization of HIF-2a and the activation of the ERYTHROPOIETIN gene, leading to expansion of red cell mass. We are still at an early stage of understanding how PHD2 works. PHD2 has two domains, a catalytic domain and a zinc finger domain. The former catalyzes prolyl hydroxylation of HIF- 2a. The function of the latter has been elusive. A critical role for the latter is evidenced by patients with erythrocytosis who harbor loss of function mutations in the zinc finger of PHD2, and by mice with inactivating mutations of the zinc finger that display erythrocytosis. Previous in vitro studies have shown the zinc finger binds to a Pro-Xaa-Leu-Glu (PXLE) motif that is found in components of the HSP90 pathway, including p23, FKBP38, HSP90a, and HSP90a, as well as in the ribosomal chaperone NACA. The HIF pathway is present in all metazoans, and this PXLE motif shows strong conservation in these proteins across metazoan species. This leads to a model in which this motif recruits PHD2 to the translation and HSP90 pathways to facilitate hydroxylation of HIF-2a, which is a known client of the protein folding HSP90 pathway. In this manner, PHD2 can maintain tight control over HIF-2a levels. The aforementioned observations on erythrocytosis identify a critical role for the zinc finger. However, they do not identify which of these PXLE-containing proteins, if any, are essential for normal regulation of red cell mass by PHD2. To address this, we have recently generated mice with knockin missense mutations that ablate the Pro-Xaa-Leu-Glu motif in the p23, Fkbp38, Hsp90a, Hsp90b, and Naca genes to prevent the interaction of the respective proteins with PHD2. We will examine the individual knockin mice as well as combinations of knockins for hemoglobin concentration, hematocrit, red blood cell count, and serum Epo levels. We will determine the tissue source of Epo mRNA. We will also cross these mice with a Tibetan Phd2 knockin mouse that bears a double amino acid substitution in its zinc finger that selectively impairs interaction with p23 in order to independently assess the importance of these interactions. The proposed studies will identify a mechanism by which patients with PHD2 zinc finger mutations develop erythrocytosis. Importantly, these studies will also resolve the apparent paradox of how high altitude-adapted Tibetans can harbor mutations that impair PHD2 zinc finger function and at the same time avoid erythrocytosis. The studies will reveal unanticipated links between oxygen sensing and the translation and HSP90 pathways in the control of red cell mass.

项目摘要/摘要 控制红细胞质量的中央氧气传感器是PHD2（也称为EGLN1）。在 肾皮质的专业间质细胞，PHD2脯氨酰羟基化转录因子HIF-2A中的HIF-2A 氧依赖性方式并将其靶向降解。在低氧条件下，羟基化羟基化 被捕，导致HIF-2A稳定和促红细胞生成基因的激活 膨胀红细胞质量。我们仍处于了解PHD2的工作原理的早期阶段。 PHD2具有 两个域，一个催化域和一个锌指域。前者催化HIF-羟基化羟基化 2a。后者的功能是难以捉摸的。患者证明了后者的关键作用 红细胞增多症患者在phd2的锌指中丧失功能突变，并由带有的小鼠 表现出红细胞增多的锌指的灭活突变。以前的体外研究表明 锌指与在HSP90途径的组件中发现的Pro-XAA-Leu-Glu（PXLE）基序结合， 包括P23，FKBP38，HSP90A和HSP90A，以及核糖体伴侣NACA。 hif 途径都存在于所有后生动物中，并且该PXLE基序在这些蛋白质中显示出强烈的保守性 跨后生动物。这导致了一个模型，该图案将phd2招募到翻译和 HSP90促进HIF-2A羟基化的途径，这是蛋白质折叠HSP90的知名客户 路径。通过这种方式，PHD2可以维持对HIF-2A水平的严格控制。 关于红细胞增多症的上述观察结果确定了锌指的关键作用。 但是，他们没有识别哪种含PXLE的蛋白质（如果有的话）对于正常 通过PHD2调节红细胞质量。为了解决这个问题，我们最近用敲门蛋白产生了老鼠 在P23，FKBP38，HSP90A，HSP90B和NACA中烧掉Pro-XAA-Leu-Glu图案的错义突变 防止各个蛋白与PHD2相互作用的基因。我们将检查个人 敲击蛋白小鼠以及敲蛋白的组合，用于血红蛋白浓度，血细胞比容，红细胞 计数和血清EPO水平。我们将确定EPO mRNA的组织来源。我们还将越过这些 用藏族PHD2敲击蛋白小鼠在其锌指中带有双氨基酸取代的小鼠 有选择地损害与P23的互动，以便独立评估这些的重要性 互动。拟议的研究将确定一种机制，phd2锌指的患者 突变会产生红细胞增多。重要的是，这些研究还将解决明显的悖论 高空适应的藏族可以藏有损害PHD2锌指功能的突变 时间避免红细胞增多。研究将揭示氧气和氧气之间的意外联系 在控制红细胞质量中的翻译和HSP90途径。