Masson N, Willam C, Maxwell P H, Pugh C W, Ratcliffe P J
The Henry Wellcome Building of Genomic Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
EMBO J. 2001 Sep 17;20(18):5197-206. doi: 10.1093/emboj/20.18.5197.
Oxygen-dependent proteolytic destruction of hypoxia-inducible factor-alpha (HIF-alpha) subunits plays a central role in regulating transcriptional responses to hypoxia. Recent studies have defined a key function for the von Hippel-Lindau tumour suppressor E3 ubiquitin ligase (VHLE3) in this process, and have defined an interaction with HIF-1 alpha that is regulated by prolyl hydroxylation. Here we show that two independent regions within the HIF-alpha oxygen-dependent degradation domain (ODDD) are targeted for ubiquitylation by VHLE3 in a manner dependent upon prolyl hydroxylation. In a series of in vitro and in vivo assays, we demonstrate the independent and non-redundant operation of each site in regulation of the HIF system. Both sites contain a common core motif, but differ both in overall sequence and in the conditions under which they bind to the VHLE3 ligase complex. The definition of two independent destruction domains implicates a more complex system of pVHL-HIF-alpha interactions, but reinforces the role of prolyl hydroxylation as an oxygen-dependent destruction signal.
缺氧诱导因子-α（HIF-α）亚基的氧依赖性蛋白水解破坏在调节对缺氧的转录反应中起核心作用。最近的研究确定了冯·希佩尔-林道肿瘤抑制因子E3泛素连接酶（VHLE3）在此过程中的关键功能，并确定了其与受脯氨酰羟化调节的HIF-1α的相互作用。在此，我们表明，HIF-α氧依赖性降解结构域（ODDD）内的两个独立区域以脯氨酰羟化依赖的方式成为VHLE3泛素化的靶点。在一系列体外和体内试验中，我们证明了每个位点在HIF系统调节中的独立且非冗余作用。两个位点都包含一个共同的核心基序，但在整体序列以及它们与VHLE3连接酶复合物结合的条件方面都有所不同。两个独立破坏结构域的确定意味着pVHL-HIF-α相互作用系统更为复杂，但强化了脯氨酰羟化作为氧依赖性破坏信号的作用。
