Wilkins Sarah E, Hyvärinen Jaana, Chicher Johana, Gorman Jeffrey J, Peet Daniel J, Bilton Rebecca L, Koivunen Peppi
School of Molecular and Biomedical Science and the Australian Research Council Special Research Centre for the Molecular Genetics of Development, University of Adelaide, Adelaide, SA 5005, Australia.
Int J Biochem Cell Biol. 2009 Jul;41(7):1563-71. doi: 10.1016/j.biocel.2009.01.005. Epub 2009 Jan 20.
FIH-1, factor inhibiting hypoxia-inducible factor-1 (HIF-1), regulates oxygen sensing by hydroxylating an asparagine within HIF-alpha. It also hydroxylates asparagines in many proteins containing ankyrin repeats, including Notch1-3, p105 and I?B?. Relative binding affinity and hydroxylation rate are crucial determinants of substrate selection and modification. We determined the contributions of substrate sequence composition and length and of oxygen concentration to the FIH-1-binding and/or hydroxylation of Notch1-4 and compared them with those for HIF-1alpha. We also demonstrated hydroxylation of two asparagines in Notch2 and 3, corresponding to Sites 1 and 2 of Notch1, by mass spectrometry for the first time. Our data demonstrate that substrate length has a much greater influence on FIH-1-dependent hydroxylation of Notch than of HIF-1alpha, predominantly through binding affinity rather than maximal reaction velocity. The K(m) value of FIH-1 for Notch1, < 0.2 microM, is at least 250-fold lower than that of 50 microM for HIF-1alpha. Site 1 of Notch1-3 appeared the preferred site of FIH-1 hydroxylation in these substrates. Interestingly, binding of Notch4 to FIH-1 was observed with an affinity almost 10-fold lower than for Notch1-3, but no hydroxylation was detected. Importantly, we demonstrate that the K(m) of FIH-1 for oxygen at the preferred Site 1 of Notch1-3, 10-19 microM, is an order of magnitude lower than that for Site 2 or HIF-1alpha. Hence, at least during in vitro hydroxylation, Notch is likely to become efficiently hydroxylated by FIH-1 even under relatively severe hypoxic conditions, where HIF-1alpha hydroxylation would be reduced.
FIH-1（缺氧诱导因子-1抑制因子）通过羟基化缺氧诱导因子-α（HIF-α）中的天冬酰胺来调节氧感应。它还能羟基化许多含有锚蛋白重复序列的蛋白质中的天冬酰胺，包括Notch1-3、p105和IκBα。相对结合亲和力和羟基化速率是底物选择和修饰的关键决定因素。我们确定了底物序列组成和长度以及氧浓度对Notch1-4与FIH-1结合和/或羟基化的贡献，并将其与HIF-1α的贡献进行了比较。我们还首次通过质谱法证实了Notch2和3中两个对应于Notch1第1和第2位点的天冬酰胺发生了羟基化。我们的数据表明，底物长度对Notch依赖FIH-1的羟基化的影响远大于对HIF-1α的影响，主要是通过结合亲和力而非最大反应速度。FIH-1对Notch1的K(m)值<0.2μM，至少比HIF-1α的50μM低250倍。在这些底物中，Notch1-3的第1位点似乎是FIH-1羟基化的首选位点。有趣的是，观察到Notch4与FIH-1的结合亲和力比Notch1-3低近10倍，但未检测到羟基化。重要的是，我们证明了FIH-1在Notch1-3的首选第1位点对氧的K(m)值为10-19μM，比第2位点或HIF-1α低一个数量级。因此，至少在体外羟基化过程中，即使在相对严重的缺氧条件下（此时HIF-1α的羟基化会减少），Notch也可能被FIH-1有效地羟基化。
