Blum Andreas, Raum Andre, Maser Edmund
Department of Experimental Toxicology, School of Medicine, University of Kiel, Brunswiker Strasse 10, D-24105 Kiel, Germany.
Biochemistry. 2003 Apr 15;42(14):4108-17. doi: 10.1021/bi027425d.
11 beta-Hydroxysteroid dehydrogenase type 1 (11 beta-HSD 1) catalyzes the interconversion of inactive into active glucocorticoids and has been shown to play a key role in metabolic disorders such as obesity and diabetes. 11 beta-HSD 1 belongs to the short chain dehydrogenases/reductases (SDR) and shares all common structural motifs typically for this protein superfamily. Unlike common SDRs, 11 beta-HSD 1 is N-terminally extended by a hydrophobic domain that anchors this enzyme in the endoplasmic reticulum (ER) membrane. Interestingly, the occurrence of 11 beta-HSD 1 transcripts lacking the N-terminal hydrophobic domain has repeatedly been reported in a variety of tissues, and the corresponding protein has been named 11 beta-HSD 1B. So far, no activity of 11 beta-HSD 1B has been observed, such that a physiological role could not be ascribed. In the present investigation, we showed for the first time that the truncated human 11 beta-HSD 1B form, expressed in the yeast Pichia pastoris, may indeed be active. However, this activity was prevented by the fact that 11 beta-HSD 1B is still kept attached to the ER membrane. Via computer assisted simulation and modeling, we identified a putative domain within the 11 beta-HSD 1 structure that could be responsible for this additional membrane attachment. By performing site-directed mutagenesis, heterologous expression, immunoblot analysis, and activity assays, we verified that this hydrophobic domain could indeed interact with the ER membrane and that some of the introduced mutations (V149R, V149E) led to a release of 11 beta-HSD 1B from membrane attachment without affecting its enzymatic activity. However, the activity of 11 beta-HSD 1B proved to be very unstable and was lost within hours after solubilization and release from the ER membrane. Importantly, 11 beta-HSD 1 constructs lacking the first 15 N-terminal amino acids and bearing additional amino acid substitutions (t15-V149R, t15-V149E) were then found to be soluble and to be stable in terms of enzyme activity. Combined, despite its occurrence in mammalian tissues, 11 beta-HSD 1B has obviously no physiological role since it is either inactive while being attached to the ER or it is rapidly losing activity once being released from intracellular membranes. Our findings with the t15-V149R and t15-V149E constructs are promising to further understand the complex mechanical and structural properties of 11 beta-HSD 1.
11β-羟基类固醇脱氢酶1型（11β-HSD 1）催化无活性糖皮质激素与活性糖皮质激素之间的相互转化，并已被证明在肥胖症和糖尿病等代谢紊乱中起关键作用。11β-HSD 1属于短链脱氢酶/还原酶（SDR），具有该蛋白质超家族典型的所有常见结构基序。与常见的SDR不同，11β-HSD 1在N端通过一个疏水结构域延伸，该结构域将这种酶锚定在内质网（ER）膜中。有趣的是，在多种组织中反复报道了缺乏N端疏水结构域的11β-HSD 1转录本的出现，相应的蛋白质被命名为11β-HSD 1B。到目前为止，尚未观察到11β-HSD 1B的活性，因此无法确定其生理作用。在本研究中，我们首次表明，在酵母毕赤酵母中表达的截短型人11β-HSD 1B形式可能确实具有活性。然而，11β-HSD 1B仍附着在内质网膜上这一事实阻止了这种活性。通过计算机辅助模拟和建模，我们在11β-HSD 1结构中确定了一个可能负责这种额外膜附着的结构域。通过进行定点诱变、异源表达、免疫印迹分析和活性测定，我们证实了这个疏水结构域确实可以与内质网膜相互作用，并且一些引入的突变（V149R、V149E）导致11β-HSD 1B从膜附着中释放出来，而不影响其酶活性。然而，11β-HSD 1B的活性被证明非常不稳定，在从内质网膜溶解和释放后数小时内就会丧失。重要的是，随后发现缺少前15个N端氨基酸并带有额外氨基酸取代的11β-HSD 1构建体（t15-V149R、t15-V149E）是可溶的，并且在酶活性方面是稳定的。综合来看，尽管11β-HSD 1B存在于哺乳动物组织中，但它显然没有生理作用，因为它在内质网附着时无活性，或者一旦从细胞内膜释放就会迅速丧失活性。我们对t15-V149R和t15-V149E构建体的研究结果有望进一步了解11β-HSD 1复杂的力学和结构特性。
