Control of Erythropoiesis by the Oxygen Sensor PHD2

项目总结/摘要 控制红细胞质量的中央氧传感器是PHD 2（也称为EGLN 1）。在 PHD2脯氨酰使转录因子HIF-2a羟基化， 以氧依赖的方式并将其作为降解的目标。在低氧条件下，脯氨酰羟基化 被阻止，导致HIF-2a的稳定和促红细胞生成素基因的激活， 红细胞团的扩张。我们仍然处于了解PHD2如何工作的早期阶段。PHD2有 两个结构域，催化结构域和锌指结构域。前者催化HIF-1 α的脯氨酰羟基化， 2a.后者的功能一直难以捉摸。后者的关键作用由以下患者证明： 红细胞增多症的小鼠，其在PHD 2的锌指中具有功能缺失突变， 使表现红细胞增多症的锌指突变失活。以前的体外研究表明， 锌指结合于在HSP 90途径的组分中发现的Pro-Xaa-Leu-Glu（PXLE）基序， 包括p23、FKBP 38、HSP 90 a和HSP 90 a，以及核糖体伴侣NACA。述hif PXLE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强保守性 在后生动物物种中。这导致了一个模型，其中该基序将PHD2招募到翻译中， 促进HIF-2a羟基化的HSP90途径，HIF-2a是HSP90蛋白折叠的已知客户 通路通过这种方式，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确定了锌指的关键作用。 然而，他们并没有确定这些含有PXLE的蛋白质中的哪一种，如果有的话，是正常发育所必需的。 PHD2对红细胞质量的调节。为了解决这一问题，我们最近制造了具有敲入基因的小鼠， 消除p23、Fkbp38、Hsp90 a、Hsp90 b和Naca中Pro-Xaa-Leu-Glu基序的错义突变 基因以防止相应蛋白质与PHD2的相互作用。我们会检查 敲入小鼠以及敲入的组合对于血红蛋白浓度、血细胞比容、红细胞 计数和血清Epo水平。我们将确定Epo mRNA的组织来源。我们还将跨越这些 在其锌指中携带双氨基酸取代的西藏Phd2基因敲入小鼠， 选择性地削弱与p23的相互作用，以便独立地评估这些的重要性。 交互.拟议的研究将确定PHD 2锌指患者 突变发展红细胞增多症。重要的是，这些研究还将解决一个明显的悖论， 适应高海拔的藏族人可能携带损害PHD2锌指功能的突变， 避免红细胞增多。这些研究将揭示氧感与心脏病之间意想不到的联系。 翻译和热休克蛋白90途径在红细胞质量的控制。

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.