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首页> 外文期刊>Molecular & cellular proteomics: MCP >Proteomics-based identification of novel factor inhibiting hypoxia-inducible factor (FIH) substrates indicates widespread asparaginyl hydroxylation of ankyrin repeat domain-containing proteins.
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Proteomics-based identification of novel factor inhibiting hypoxia-inducible factor (FIH) substrates indicates widespread asparaginyl hydroxylation of ankyrin repeat domain-containing proteins.

机译:基于蛋白质组学的新型因子抑制缺氧诱导因子(FIH)底物的鉴定表明含锚蛋白重复域的蛋白质普遍存在天冬酰胺基羟基化。

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摘要

Post-translational hydroxylation has been considered an unusual modification on intracellular proteins. However, following the recognition that oxygen-sensitive prolyl and asparaginyl hydroxylation are central to the regulation of the transcription factor hypoxia-inducible factor (HIF), interest has centered on the possibility that these enzymes may have other substrates in the proteome. In support of this certain ankyrin repeat domain (ARD)-containing proteins, including members of the IkappaB and Notch families, have been identified as alternative substrates of the HIF asparaginyl hydroxylase factor inhibiting HIF (FIH). Although these findings imply a potentially broad range of substrates for FIH, the precise extent of this range has been difficult to determine because of the difficulty of capturing transient enzyme-substrate interactions. Here we describe the use of pharmacological "substrate trapping" together with stable isotope labeling by amino acids in cell culture (SILAC) technology to stabilize and identify potential FIH-substrate interactions by mass spectrometry. To pursue these potential FIH substrates we used conventional data-directed tandem MS together with alternating low/high collision energy tandem MS to assign and quantitate hydroxylation at target asparaginyl residues. Overall the work has defined 13 new FIH-dependent hydroxylation sites with a degenerate consensus corresponding to that of the ankyrin repeat and a range of ARD-containing proteins as actual and potential substrates for FIH. Several ARD-containing proteins were multiply hydroxylated, and detailed studies of one, Tankyrase-2, revealed eight sites that were differentially sensitive to FIH-catalyzed hydroxylation. These findings indicate that asparaginyl hydroxylation is likely to be widespread among the approximately 300 ARD-containing species in the human proteome.
机译:翻译后羟基化被认为是对细胞内蛋白质的不寻常修饰。但是,在认识到氧敏感性脯氨酰和天冬酰胺基羟基化是转录因子低氧诱导因子(HIF)调节的核心之后,人们开始关注这些酶在蛋白质组中可能具有其他底物的可能性。为了支持这种含锚蛋白重复域(ARD)的蛋白质,包括IkappaB和Notch家族的成员,已被确定为HIF天冬酰胺基羟化酶抑制HIF(FIH)的替代底物。尽管这些发现暗示了FIH底物的潜在范围很广,但由于难以捕获瞬时酶与底物的相互作用,因此很难确定该范围的确切范围。在这里,我们描述了通过细胞培养(SILAC)技术中的氨基酸进行药理学“底物捕获”和稳定同位素标记的方法,以通过质谱稳定和识别潜在的FIH-底物相互作用。为了追踪这些潜在的FIH底物,我们使用了传统的数据定向串联质谱仪和交替的低/高碰撞能量串联质谱仪来对目标天冬酰胺基残基处的羟基化进行分配和定量。总的来说,这项工作已经定义了13个新的FIH依赖性羟基化位点,其简并性对应于锚蛋白重复序列​​的简并性,并且一系列含ARD的蛋白质是FIH的实际和潜在底物。几个含有ARD的蛋白质被多重羟基化,对其中一种(Tankyrase-2)的详细研究显示,八个位点对FIH催化的羟基化反应具有不同的敏感性。这些发现表明,人蛋白质组中大约300种含ARD的物种中,天冬酰胺基羟基化很可能广泛。

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