Appropriate function of 11β-hydroxysteroid dehydrogenase type 1 in the endoplasmic reticulum lumen is dependent on its N-terminal region sharing similar topological determinants with 50-kDa esterase
Appropriate function of 11β-hydroxysteroid dehydrogenase type 1 in the endoplasmic reticulum lumen is dependent on its N-terminal region sharing similar topological determinants with 50-kDa esterase
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DOI：
10.1074/jbc.m313666200
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发表时间：
2004-07-23
影响因子：
4.8
通讯作者：
Odermatt, A
中科院分区：
文献类型：
--
作者：
Frick, C;Atanasov, AG;Odermatt, A
通过糖皮质激素的相互转化，1 型 11β-羟基类固醇脱氢酶 (11β-HSD1) 发挥重要的前受体功能，目前被认为是一个有前途的治疗靶点。此外，11β-HSD1 在 7-酮胆固醇代谢中发挥潜在作用。在这里，我们研究了 N 末端区域对酶活性的作用，并解决了 11beta-HSD1 方向进入内质网 (ER) 腔的相关性。先前的研究表明，11β-HSD1 和 50-kDa 酯酶/芳基乙酰胺脱乙酰酶 (E3) 的管腔方向是由它们高度相似的 N 端跨膜结构域决定的。在 11beta-HSD1 中，Lys(5) 被 Ser 取代，但 E3 中类似的 Lys(4) 没有被 Ile 取代，导致 ER 膜中出现倒置拓扑，表明存在第二个拓扑决定因素。在这里，我们确定 11beta-HSD1 中的 Glu(25)/Glu(26) 和 E3 中的 Asp(25) 作为管腔方向的第二个决定因素。我们的结果表明，特定残基的确切位置而不是螺旋两侧的净电荷分布对于膜拓扑至关重要。对 N 端结构域中带电残基的分析揭示了 Lys(35)/Lys(36) 和 Glu(25)/Glu(26) 对酶活性的重要作用，表明这些残基是观察到的 N 端膜锚定对 11beta-HSD1 催化结构域的稳定作用的原因。此外，对表达野生型 11β-HSD1（面向 ER 腔）的完整细胞或突变型 K5S/K6S（面向细胞质）的活性测量表明，腔方向对于皮质醇的有效氧化至关重要。此外，我们证明 11beta-HSD1（而不是具有细胞质取向的突变体 K5S/K6S）催化 7-酮胆固醇的氧化还原。具有催化部分胞质取向的 11beta-HSD1 和 E3 构建体应该在未来解决这些蛋白质生理功能的研究中有用。
By interconverting glucocorticoids, 11beta-hydroxysteroid dehydrogenase type 1 ( 11beta-HSD1) exerts an important pre-receptor function and is currently considered a promising therapeutic target. In addition, 11beta-HSD1 plays a potential role in 7-ketocholesterol metabolism. Here we investigated the role of the N-terminal region on enzymatic activity and addressed the relevance of 11beta-HSD1 orientation into the endoplasmic reticulum ( ER) lumen. Previous studies revealed that the luminal orientation of 11beta-HSD1 and 50-kDa esterase/ arylacetamide deacetylase (E3) is determined by their highly similar N-terminal transmembrane domains. Substitution of Lys(5) by Ser in 11beta-HSD1, but not of the analogous Lys(4) by Ile in E3, led to an inverted topology in the ER membrane, indicating the existence of a second topological determinant. Here we identified Glu(25)/Glu(26) in 11beta-HSD1 and Asp(25) in E3 as the second determinant for luminal orientation. Our results suggest that the exact location of specific residues rather than net charge distribution on either side of the helix is critical for membrane topology. Analysis of charged residues in the N-terminal domain revealed an essential role of Lys(35)/Lys(36) and Glu(25)/Glu(26) on enzymatic activity, suggesting that these residues are responsible for the observed stabilizing effect of the N-terminal membrane anchor on the catalytic domain of 11beta-HSD1. Moreover, activity measurements in intact cells expressing wildtype 11beta-HSD1, facing the ER lumen, or mutant K5S/ K6S, facing the cytoplasm, revealed that the luminal orientation is essential for efficient oxidation of cortisol. Furthermore, we demonstrate that 11beta-HSD1, but not mutant K5S/ K6S with cytoplasmic orientation, catalyzes the oxoreduction of 7-ketocholesterol. 11beta-HSD1 and E3 constructs with cytosolic orientation of their catalytic moiety should prove useful in future studies addressing the physiological function of these proteins.