Asparagine and aspartate hydroxylation of the cytoskeletal ankyrin family is catalyzed by factor-inhibiting hypoxia-inducible factor.
Asparagine and aspartate hydroxylation of the cytoskeletal ankyrin family is catalyzed by factor-inhibiting hypoxia-inducible factor.
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DOI：
10.1074/jbc.m110.193540
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发表时间：
2011-03-04
期刊：
影响因子：
--
通讯作者：
Schofield CJ
中科院分区：
文献类型：
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作者：
Yang M;Ge W;Chowdhury R;Claridge TD;Kramer HB;Schmierer B;McDonough MA;Gong L;Kessler BM;Ratcliffe PJ;Coleman ML;Schofield CJ
因子抑制缺氧诱导因子（FIH）催化缺氧诱导因子（HIF）C-末端转录激活结构域中天冬酰胺残基的β-羟基化，这是一种负调节HIF转录活性的修饰。FIH还催化普遍存在的含锚蛋白重复结构域（ARD）的蛋白质内高度保守的Asn残基的羟基化。在三重复共有锚蛋白的情况下，羟基化已被证明可以稳定ARD折叠的局部区域，但这种现象尚未在广泛的天然存在的ARD中得到证实。在这里，我们报告说，细胞骨架锚蛋白家族的FIH催化的羟基化的底物。我们发现，ARD的ankeplatin R是多羟基化的FIH在体外和内源性蛋白纯化的人和小鼠红细胞。锚蛋白ARD（锚蛋白重复序列13-24）的D34区域的羟基化增加了其构象稳定性，并导致其与带3（CDB 3）的胞质结构域的相互作用减少，证明了FIH催化的羟基化调节蛋白质-蛋白质相互作用的潜力。出乎意料的是，我们发现，天冬氨酸残基在anke 3R和anke 3B的羟基化和FIH催化的天冬氨酸羟基化也发生在其他天然存在的AR序列。与Asp-底物肽复合的FIH变体的晶体结构以及羟基化产物的NMR分析鉴定了FIH催化的Asp羟基化的3S区域和立体选择性，揭示了以前前所未有的翻译后修饰。
Factor-inhibiting hypoxia-inducible factor (FIH) catalyzes the β-hydroxylation of an asparagine residue in the C-terminal transcriptional activation domain of the hypoxia inducible factor (HIF), a modification that negatively regulates HIF transcriptional activity. FIH also catalyzes the hydroxylation of highly conserved Asn residues within the ubiquitous ankyrin repeat domain (ARD)-containing proteins. Hydroxylation has been shown to stabilize localized regions of the ARD fold in the case of a three-repeat consensus ankyrin protein, but this phenomenon has not been demonstrated for the extensive naturally occurring ARDs. Here we report that the cytoskeletal ankyrin family are substrates for FIH-catalyzed hydroxylations. We show that the ARD of ankyrinR is multiply hydroxylated by FIH both in vitro and in endogenous proteins purified from human and mouse erythrocytes. Hydroxylation of the D34 region of ankyrinR ARD (ankyrin repeats 13–24) increases its conformational stability and leads to a reduction in its interaction with the cytoplasmic domain of band 3 (CDB3), demonstrating the potential for FIH-catalyzed hydroxylation to modulate protein-protein interactions. Unexpectedly we found that aspartate residues in ankyrinR and ankyrinB are hydroxylated and that FIH-catalyzed aspartate hydroxylation also occurs in other naturally occurring AR sequences. The crystal structure of an FIH variant in complex with an Asp-substrate peptide together with NMR analyses of the hydroxylation product identifies the 3S regio- and stereoselectivity of the FIH-catalyzed Asp hydroxylation, revealing a previously unprecedented posttranslational modification.