Control of Erythropoiesis by the Oxygen Sensor PHD2

通过氧传感器 PHD2 控制红细胞生成

基本信息

批准号：
10618878
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
项目类别：
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/10618878
关键词：
Ablation Address Alleles Altitude Amino Acid Substitution Anemia Binding Blood C-terminal Catalytic Domain Cell Count Client Clinical Treatment Complex Curiosities Erythrocyte Indices Erythrocytes Erythrocytoses 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 Variant Work Zinc Fingers insight interest kidney cortex loss of function mutation polypeptide prevent protein folding recruit sensor transcription factor

项目摘要

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.

项目概要/摘要控制红细胞质量的中枢氧传感器是 PHD2（也称为 EGLN1）。在肾皮质特化间质细胞中，PHD2 脯氨酰羟化转录因子 HIF-2a 一种依赖于氧的方式并针对其进行降解。缺氧条件下，脯氨酰羟基化被抑制，导致 HIF-2a 稳定并激活促红细胞生成素基因，从而导致红细胞团的扩张。我们仍处于了解 PHD2 工作原理的早期阶段。 PHD2有两个结构域，一个催化结构域和一个锌指结构域。前者催化HIF-的脯氨酰羟基化 2a.后者的功能一直难以捉摸。患有以下疾病的患者证明了后者的关键作用携带 PHD2 锌指功能缺失突变的红细胞增多症，以及患有红细胞增多症的小鼠显示红细胞增多症的锌指失活突变。先前的体外研究表明锌指与 HSP90 通路组件中发现的 Pro-Xaa-Leu-Glu (PXLE) 基序结合，包括 p23、FKBP38、HSP90a 和 HSP90a，以及核糖体伴侣 NACA。高强度IF 途径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很强的保守性跨后生动物物种。这导致了一个模型，其中该基序招募 PHD2 进行翻译并 HSP90 途径促进 HIF-2a 羟基化，HIF-2a 是蛋白质折叠 HSP90 的已知客户途径。通过这种方式，PHD2 可以保持对 HIF-2a 水平的严格控制。上述对红细胞增多症的观察表明锌指的关键作用。然而，他们并没有确定这些含有 PXLE 的蛋白质（如果有的话）中的哪些对于正常的生命活动是必需的。 PHD2 调节红细胞质量。为了解决这个问题，我们最近培育了具有基因敲入的小鼠错义突变消除 p23、Fkbp38、Hsp90a、Hsp90b 和 Naca 中的 Pro-Xaa-Leu-Glu 基序基因来阻止各自的蛋白质与 PHD2 相互作用。我们将检查个人基因敲入小鼠以及血红蛋白浓度、血细胞比容、红细胞的基因敲入组合计数和血清 Epo 水平。我们将确定 Epo mRNA 的组织来源。我们也会跨越这些带有西藏 Phd2 敲入小鼠的小鼠，该小鼠的锌指具有双氨基酸取代，选择性损害与 p23 的相互作用，以便独立评估这些相互作用的重要性互动。拟议的研究将确定 PHD2 锌指患者的机制突变会导致红细胞增多症。重要的是，这些研究还将解决如何解决这个明显的悖论。适应高海拔的藏族人可能携带损害 PHD2 锌指功能的突变及时避免红细胞增多。这些研究将揭示氧传感和氧传感器之间意想不到的联系翻译和 HSP90 通路在红细胞质量控制中的作用。