Control of Erythropoiesis by the Oxygen Sensor PHD2

项目摘要/摘要 控制红细胞质量的中枢氧传感器是PHD2(也称为EGLN1)。在……里面 肾皮质的特化间质细胞，PHD2，Pro羟基化转录因子HIF-2a在 一种依赖氧气的方式，并将其作为降解的目标。在低氧条件下，脯氨酸羟化 被抑制，导致HIF-2a的稳定和促红细胞生成素基因的激活，导致 导致红细胞团的扩张。我们仍处于了解PHD2如何工作的早期阶段。PHD2有 两个结构域，一个催化结构域和一个锌指结构域。前者催化HIF-的Pro-羟基化。 2A。后者的作用一直难以捉摸。后者的关键作用由以下患者证明 存在PHD2锌指功能突变的红细胞增多症小鼠 显示红细胞增多的锌指的失活突变。此前的体外研究表明， 锌指与一个Pro-xaa-Leu-Glu(PXLE)基序结合，该基序存在于HSP90途径的组成部分中， 包括p23、FKBP38、HSP90a和HSP90a，以及核糖体伴侣NACA。高密度脂蛋白 在所有后生动物中都存在途径，并且这个PXLE基序在这些蛋白质中显示出很强的保守性 横跨后生动物物种。这导致了一个模型，在该模型中，该母题招募PHD2进行翻译，并 HSP90促进HIF-2a羟化的途径，HIF-2a是蛋白质折叠HSP90的已知客户 路径。通过这种方式，PHD2可以保持对HIF-2a水平的严格控制。 前述对红细胞增多症的观察确定了锌指的关键作用。 然而，他们没有确定这些含有PXLE的蛋白质中哪些是正常所必需的 PHD2对红细胞质量的调节作用。为了解决这个问题，我们最近培育出了敲门声小鼠 破坏p23、Fkbp38、Hsp90a、Hsp90b和NacA中Pro-xaa-Leu-Glu基序的错义突变 防止各自的蛋白质与PHD2相互作用的基因。我们将检查个人 Knockin小鼠及其组合对血红蛋白浓度、红细胞压积、红细胞数的影响 计数和血清促红细胞生成素水平。我们将确定EPO mRNA的组织来源。我们也会穿过这些 一只西藏PHD2敲击小鼠的小鼠，它的锌手指上有双氨基酸取代， 选择性地损害与p23的相互作用，以便独立评估这些 互动。拟议的研究将确定PHD2锌指患者的机制 突变会导致红细胞增多症。重要的是，这些研究还将解决一个明显的悖论： 高原适应的藏族人可以携带损害PHD2锌指功能的突变，同时 及时避免红细胞增多。这些研究将揭示氧气传感和 翻译和HSP90途径在控制红细胞质量中的作用。

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.