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：
9146882
负责人：
FRANK S LEE
金额：
$ 36万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-21 至 2020-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9146882
关键词：
Address Altitude Amino Acid Substitution Anemia Attenuated Binding Binding Sites Catalytic Domain Cells Chronic Client Dependency Disease Erythrocytes Erythrocytoses Erythroid Erythropoiesis Event Gene Expression Gene Targeting Genes Genetic Transcription Genetic study Hearing Impaired Persons Heat-Shock Proteins 90 Homeostasis Hydroxylation Hypersensitivity Hypoxia Hypoxia Inducible Factor Kidney Knock-in Mouse Knowledge Left Mediating Modeling Modification Molecular Mus Mutagenesis Mutate Myelogenous Nature Oxygen Pathway interactions Procollagen-Proline Dioxygenase Protein Isoforms Proteins Reaction Recruitment Activity Red Cell Mass result Regulation Response Elements Role Signaling Protein Staging Tertiary Protein Structure Testing Therapeutic Tissues Translating Up-Regulation VHL protein Variant Zinc Zinc Fingers abstracting base bone flexibility kidney cell novel progenitor receptor research study sensor transcription factor

项目摘要

PROJECT SUMMARY/ABSTRACT 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-2α (HIF-2α). When oxygen is plentiful, constitutive hydroxylation of HIF-2α by PHD2 targets the former for degradation. When oxygen is lacking, PHD2 activity is attenuated, leading to the stabilization of HIF-2α 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-α's (of which HIF-1α and HIF-2α 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-α 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-1α and Hif-2α heterozygous deficient mice to determine Hif-α 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.

项目概要/摘要将氧浓度变化转化为红细胞质量变化的关键事件是脯氨酰羟化酶结构域蛋白 2 (PHD2) 催化转录因子的羟基化，缺氧诱导因子 2α (HIF-2α) 当氧气充足时，PHD2 对 HIF-2α 进行组成性羟基化。当缺氧时，PHD2 活性减弱，导致稳定。 HIF-2α 和肾脏中循环 EPO 基因的激活结合。我们对 PHD2 的了解仍处于初级阶段。 PHD2 包含两个进化保守的结构域，一个脯氨酰羟化酶结构域，催化羟基化反应，而预测的锌指结构域人们知之甚少。值得注意的是，我们最近发现的其他两种哺乳动物 PHD 旁系同源物缺乏后者。发现髓样神经聋 (MYND) 类型的锌指结构域与高对 HSP90 途径的选定辅助伴侣中存在的 Pro-Xaa-Leu-Glu 基序的严格性，例如 HIF-α（其中 HIF-1α 和 HIF-2α 是主要亚型）是 HSP90 的客户蛋白。途径，因此这导致了一个模型，其中 p23 将 PHD2 募集到 HSP90 以促进 HIF-α 因此，PHD2 的锌指具有积极的调节作用。悬而未决的问题是 PHD2 的锌指是否实际上是 MYND 型，以及它是否功能与红细胞生成有关，更一般地说，与氧稳态有关。我们建议通过实现以下具体目标 1 来解决这些问题。我们将对 PHD2 的锌指进行点突变研究，以确定是否存在可能的残基根据与已知 MYND 锌指的同源性预测其功能上很重要，确实很重要在特定目标 2 中，我们将表征与 p23 的 Pro-Xaa-Leu-Glu 基序的相互作用。小鼠系，其中两个预测的锌螯合残基已发生突变（C36S/C42S），以便我们将检查该小鼠的红细胞生成失调，并将其与锌指功能进行杂交。 Hif-1α 和 Hif-2α 杂合缺陷小鼠以确定 Hif-α 依赖性。在特定目标 3 中，我们将确定从这些小鼠获得的红系祖细胞是否对 Epo 表现出过敏，这表明该锌指具有与 Epo 无关的作用，与 Epo 基因的调节不同我们还将检查这些组织中 Hif 靶基因是否存在更广泛的上调。总的来说，我们预计这些研究将为 PHD2（一种关键信号传导）提供关键信息。调节红细胞质量的蛋白质。