Control of Erythropoiesis by the Oxygen Sensor PHD2

项目总结/摘要 脯氨酰是将氧浓度变化转换为红细胞质量变化的关键事件 羟化酶结构域蛋白2（PHD 2）催化的缺氧诱导转录因子羟化 因子-2 α（HIF-2α）。当氧充足时，PHD 2对HIF-2α的组成性羟基化作用靶向HIF-2α的表达。 前降解。当缺氧时，PHD 2活性减弱，导致PHD 2的稳定。 HIF-2α和肾脏中促红细胞生成素（EPO）基因的激活。循环EPO然后结合 红细胞祖细胞上的EPO受体，以扩大红细胞群。我们对PHD 2的理解仍然处于 非常早期的阶段。PHD 2包含两个进化上保守的结构域，一个脯氨酰羟化酶结构域， 催化羟基化反应，以及预测的锌指结构域。关于这一点，我们知之甚少。 后者，其中，可以注意到，是缺乏在其他两个哺乳动物PHD旁系同源。我们最近 发现锌指结构域，这是髓样神经性耳聋（MYND）型，结合高 对存在于HSP 90途径的选择辅伴侣中的Pro-Xaa-Leu-Glu基序的严格性， P23的。HIF-α（其中HIF-1α和HIF-2α是主要的同种型）是HSP 90的客户蛋白， 因此，这导致了一个模型，其中p23招募PHD 2到HSP 90，以促进HIF-α 羟基化因此，PHD 2的锌指被认为具有积极的调节作用。关键 尚未回答的问题是PHD 2的锌指是否实际上是MYND型，以及它是否是MYND型。 功能与红细胞生成有关，更一般地，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。具体目标1、 我们将对PHD 2的锌指进行点突变研究，以确定可能被 基于与已知MYND锌指的同源性预测为功能重要的锌指确实重要 用于与p23的Pro-Xaa-Leu-Glu基序相互作用。在具体目标2中，我们将描述敲入 小鼠系，其中两个预测的锌螯合残基已经突变（C36 S/C42 S）， 消融锌指功能。我们将检查这只小鼠的红细胞生成失调，并将其与 Hif-1α和Hif-2α杂合缺陷小鼠，以确定Hif-α依赖性。在具体目标3中，我们 确定从这些小鼠中获得的红系祖细胞是否对Epo表现出超敏反应， 这将指出，这种锌指的作用与Epo基因的调控不同， 本身我们还将检查这些组织中是否有更广泛的Hif靶基因上调， 小鼠总的来说，我们预计这些研究将为PHD 2提供关键信息，PHD 2是一个关键的信号转导途径。 调节红细胞质量的蛋白质。

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.