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: 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
• 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

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.

项目摘要/摘要 将氧气浓度变化转变为红细胞质量变化的关键事件是prolyl 羟化酶结构域蛋白2（PHD2）催化的转录因子的羟基化，低氧诱导 因子2α（HIF-2α）。当氧气丰富时，PHD2对HIF-2α的构型羟基化目标 以前的退化。当缺乏氧气时，PHD2活性会减弱，导致稳定 HIF-2α和肾脏中红细胞生成素（EPO）基因的激活。然后循环EPO结合 向红细胞祖细胞上的EPO接收器扩大红细胞质量。我们对PHD2的理解仍在 很早的阶段。 PHD2包含两个进化保守的结构域，一个丙酰羟化酶结构域，该结构域 催化羟基化反应和预测的锌指域。关于 后者，可能会注意到，其他两个哺乳动物的博士旁系同源物缺乏。我们最近有 发现髓样神经聋（MYND）类型的锌指域与高 对HSP90途径的某些联合酮中存在的Pro-XAA-Leu-Glu基序的严格性 作为p23。 HIF-α（HIF-1α和HIF-2α是主要同工型）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 羟基化。这是提出PHD2的锌指的作用，以发挥积极的调节作用。钥匙 未解决的问题是PHD2的锌指实际上是Mynd类型的，以及它是否是 功能与红细胞生成有关，更普遍地是氧稳态。 我们建议通过追求以下特定目标来解决这些问题。在特定的目标1中， 我们将在PHD2的锌指的锌指中进行点诱变研究，以确定可能是退休 预计基于与已知Mynd锌手指的同源性具有功能很重要确实很重要 用于与P23的Pro-XAA-Leu-Glu基序相互作用。在特定的目标2中，我们将描述敲击蛋白 小鼠系列已突变的两个预测的锌螯合残差（C36S/C42S） 消融锌指功能。我们将检查该鼠标是否有促红细胞生成的失调，并与之交叉 HIF-1α和HIF-2α杂合缺乏小鼠以确定HIF-α依赖性。在特定的目标3中，我们将 确定从这些小鼠获得的红细胞祖细胞是否表现出对EPO的超敏反应， 将指出该锌指的EPO独立作用，与EPO基因的调节不同 本身。我们还将检查这些组织中HIF靶基因的上调是否更广泛 老鼠。总体而言，我们预计这些研究将为PHD2提供关键信息，PHD2是一个关键的信号传导 调节红细胞肿块的蛋白质。