Furlow Paul W, Percy Melanie J, Sutherland Scott, Bierl Charlene, McMullin Mary Frances, Master Stephen R, Lappin Terence R J, Lee Frank S
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 2009 Apr 3;284(14):9050-8. doi: 10.1074/jbc.M808737200. Epub 2009 Feb 10.
A classic physiologic response to hypoxia in humans is the up-regulation of the ERYTHROPOIETIN (EPO) gene, which is the central regulator of red blood cell mass. The EPO gene, in turn, is activated by hypoxia inducible factor (HIF). HIF is a transcription factor consisting of an alpha subunit (HIF-alpha) and a beta subunit (HIF-beta). Under normoxic conditions, prolyl hydroxylase domain protein (PHD, also known as HIF prolyl hydroxylase and egg laying-defective nine protein) site specifically hydroxylates HIF-alpha in a conserved LXXLAP motif (where underlining indicates the hydroxylacceptor proline). This provides a recognition motif for the von Hippel Lindau protein, a component of an E3 ubiquitin ligase complex that targets hydroxylated HIF-alpha for degradation. Under hypoxic conditions, this inherently oxygen-dependent modification is arrested, thereby stabilizing HIF-alpha and allowing it to activate the EPO gene. We previously identified and characterized an erythrocytosis-associated HIF2A mutation, G537W. More recently, we reported two additional erythrocytosis-associated HIF2A mutations, G537R and M535V. Here, we describe the functional characterization of these two mutants as well as a third novel erythrocytosis-associated mutation, P534L. These mutations affect residues C-terminal to the LXXLAP motif. We find that all result in impaired degradation and thus aberrant stabilization of HIF-2alpha. However, each exhibits a distinct profile with respect to their effects on PHD2 binding and von Hippel Lindau interaction. These findings reinforce the importance of HIF-2alpha in human EPO regulation, demonstrate heterogeneity of functional defects arising from these mutations, and point to a critical role for residues C-terminal to the LXXLAP motif in HIF-alpha.
人类对缺氧的一种经典生理反应是促红细胞生成素（EPO）基因的上调，该基因是红细胞量的核心调节因子。反过来，EPO基因由缺氧诱导因子（HIF）激活。HIF是一种转录因子，由一个α亚基（HIF-α）和一个β亚基（HIF-β）组成。在常氧条件下，脯氨酰羟化酶结构域蛋白（PHD，也称为HIF脯氨酰羟化酶和产卵缺陷九蛋白）在保守的LXXLAP基序（下划线表示羟基接受脯氨酸）中对HIF-α进行位点特异性羟基化。这为冯·希佩尔·林道蛋白提供了一个识别基序，冯·希佩尔·林道蛋白是E3泛素连接酶复合物的一个组成部分，该复合物将羟基化的HIF-α靶向降解。在缺氧条件下，这种固有的氧依赖性修饰被阻断，从而使HIF-α稳定，并使其能够激活EPO基因。我们之前鉴定并表征了一种与红细胞增多症相关的HIF2A突变，G537W。最近，我们又报道了另外两种与红细胞增多症相关的HIF2A突变，G537R和M535V。在这里，我们描述了这两种突变体以及第三种新的与红细胞增多症相关的突变P534L的功能特征。这些突变影响LXXLAP基序C端的残基。我们发现所有这些突变都会导致降解受损，从而导致HIF-2α异常稳定。然而，它们在对PHD2结合和冯·希佩尔·林道相互作用的影响方面表现出不同的特征。这些发现强化了HIF-2α在人类EPO调节中的重要性，证明了这些突变导致的功能缺陷的异质性，并指出了LXXLAP基序C端残基在HIF-α中的关键作用。
