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 将氧浓度的变化转换为红细胞质量的变化的一个关键事件是脯氨酸基。 羟基酶结构域蛋白2(PHD2)催化的转录因子羟化，缺氧诱导 2因子α(HIF-2α)。当氧气充足时，PHD2对缺氧诱导因子-2α的结构性羟化作用针对 前者代表降级。缺氧时，PHD2活性减弱，导致PHD2活性稳定 缺氧诱导因子-2α与肾脏促红细胞生成素基因的激活。然后循环中的促红细胞生成素结合 使红细胞祖细胞上的EPO受体扩大红细胞团。我们对PHD2的理解还处于一个阶段 非常早期的阶段。PHD2包含两个进化保守的结构域，一个Pro羟基酶结构域， 催化羟化反应，并预测锌指结构域。人们对此知之甚少 后者，可以注意到，这是缺乏在其他两个哺乳动物博士类似物。我们最近做了 发现锌指结构域属于髓样神经性耳聋(Mynd)类型，它与高水平的 对HSP90途径的精选辅伴侣中存在的Pro-xaa-Leu-Glu基序的严密性，如 作为p23。HIF-α的S(其中HIF-1α和HIF-2α是其主要亚型)是热休克蛋白90的客户蛋白 途径，这因此导致了一种模型，在该模型中，p23招募PHD2到HSP90以促进HIF-α 羟化作用。因此，PHD2的锌指被认为具有积极的调节作用。钥匙 悬而未决的问题是，PHD2的锌指是否真的是mynd型的，以及它的 功能与红细胞生成有关，更广泛地说，与氧稳态有关。 我们建议通过追求以下具体目标来解决这些问题。在具体目标1中， 我们将对PHD2的锌指进行点突变研究，以确定可能存在的残基 根据与已知的myd和锌指同源性预测的功能重要性确实很重要 用于与p23的Pro-xaa-Leu-Glu基序相互作用。在具体目标2中，我们将描述敲门声 两个预测的锌螯合残基(C36S/C42S)发生突变的小鼠品系 消融锌指功能。我们将检查这只小鼠的红细胞生成失调情况，并将其与 Hif-1α和Hif-2α杂合缺陷小鼠以确定对Hif-α的依赖。在具体目标3中，我们将 确定从这些小鼠获得的红系祖细胞是否对EPO表现出超敏反应， 将表明这种锌指具有不依赖于EPO的作用，这与EPO基因的调控不同 它本身。我们还将研究在这些组织中是否存在更广泛的HIF靶基因上调 老鼠。总的来说，我们预计这些研究将为PHD2，一种关键的信号转导提供关键信息 调节红细胞质量的蛋白质。