Quantitative Mass Spectrometry Reveals Dynamics of Factor-inhibiting Hypoxia-inducible Factor-catalyzed Hydroxylation
Quantitative Mass Spectrometry Reveals Dynamics of Factor-inhibiting Hypoxia-inducible Factor-catalyzed Hydroxylation
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DOI：
10.1074/jbc.m111.262808
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发表时间：
2011-09-30
影响因子：
4.8
通讯作者：
Cockman, Matthew E.
中科院分区：
文献类型：
--
作者：
Singleton, Rachelle S.;Trudgian, David C.;Cockman, Matthew E.
天冬酰胺羟基酶是一种抑制缺氧诱导因子(HIF)的因子，是控制HIF活性的氧感应通路的中心。因子抑制缺氧诱导因子(FIH)还催化一大组蛋白质的羟化，这些蛋白质共享一个称为Ankyrin Repeat结构域(ARD)的结构基序。体外研究已经确定了FIH在不同底物上的动力学特性，并表明FIH与某些ARD蛋白的结合比HIF更紧密，ARD羟化可能具有比HIF羟化更低的氧的K-m值。然而，在细胞中对ARD底物上天冬酰胺羟基化的调节还没有系统的研究。为了解决这些问题，我们使用同位素标记和质谱学来监测在特定条件下羟化的发生、抑制和衰变。在所考察的条件下，羟化不是逆转的，而是随着蛋白质的老化而增加。添加抗坏血酸可以增加ARD蛋白的羟化程度，而补充铁和2-羟基戊二酸对ARD蛋白的羟化程度没有显著影响。尽管FIH在体外优先与ARD底物结合，但当在细胞内以融合蛋白的形式表达时，发现HIF多肽上的羟基化比ARD内的位置更完整。此外，分级缺氧条件下羟化作用的比较研究表明，ARD羟化作用受到部位特异性抑制，并且与HIF一样对缺氧抑制敏感。这些发现表明，HIF-1和ARD蛋白的天冬酰胺羟基化在相似的范围内受到氧的调节，可能通过两种底物之间的竞争来调节HIF的转录反应。
The asparaginyl hydroxylase, factor-inhibiting hypoxia-inducible factor (HIF), is central to the oxygen-sensing pathway that controls the activity of HIF. Factor-inhibiting HIF (FIH) also catalyzes the hydroxylation of a large set of proteins that share a structural motif termed the ankyrin repeat domain (ARD). In vitro studies have defined kinetic properties of FIH with respect to different substrates and have suggested FIH binds more tightly to certain ARD proteins than HIF and that ARD hydroxylation may have a lower K-m value for oxygen than HIF hydroxylation. However, regulation of asparaginyl hydroxylation on ARD substrates has not been systematically studied in cells. To address these questions, we employed isotopic labeling and mass spectrometry to monitor the accrual, inhibition, and decay of hydroxylation under defined conditions. Under the conditions examined, hydroxylation was not reversed but increased as the protein aged. The extent of hydroxylation on ARD proteins was increased by addition of ascorbate, whereas iron and 2-oxoglutarate supplementation had no significant effect. Despite preferential binding of FIH to ARD substrates in vitro, when expressed as fusion proteins in cells, hydroxylation was found to be more complete on HIF polypeptides compared with sites within the ARD. Furthermore, comparative studies of hydroxylation in graded hypoxia revealed ARD hydroxylation was suppressed in a site-specific manner and was as sensitive as HIF to hypoxic inhibition. These findings suggest that asparaginyl hydroxylation of HIF-1 and ARD proteins is regulated by oxygen over a similar range, potentially tuning the HIF transcriptional response through competition between the two types of substrate.