A steroidogenic pathway for sulfonated steroids: The metabolism of pregnenolone sulfate
A steroidogenic pathway for sulfonated steroids: The metabolism of pregnenolone sulfate
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DOI：
10.1016/j.jsbmb.2014.07.005
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发表时间：
2014-10
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影响因子：
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通讯作者：
J. Neunzig;A. Sánchez-Guijo;A. Mosa;Michaela F. Hartmann;Joachim Geyer;Stefan A. Wudy;Rita Bernhardt
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作者：
J. Neunzig;A. Sánchez-Guijo;A. Mosa;Michaela F. Hartmann;Joachim Geyer;Stefan A. Wudy;Rita Bernhardt
在许多组织中，磺化类固醇的浓度超过了游离类固醇的浓度，最近它们也被证明具有重要的生理功能。虽然先前已证明硫酸胆固醇 (CS) 可被 CYP11A1 转化为硫酸孕烯醇酮 (PregS)，但尚未详细研究 PregS 的进一步转化。为了研究磺化类固醇的类固醇生成途径是否存在类似于游离类固醇的类固醇生成途径，我们在体外重建系统中检查了 PregS 与 CYP17A1 的相互作用。差异光谱显示 CYP17A1-PregS 复合物的 Kd 值为 74.8 ± 4.2 μM，比 CYP17A1-孕烯醇酮 (Preg) 复合物高 2.5 倍。质谱实验首次证明PregS在C17位被CYP17A1羟基化，与孕烯醇酮相同。与 Preg 相比，使用 PregS 的 CYP17A1 观察到较高的 Km 值和较低的 kcat 值，表明使用磺化类固醇时催化效率降低了 40%。此外，我们分析了细胞色素b5(b5)的存在是否对PregS的CYP17A1依赖性转化有影响，正如Preg所证明的那样。有趣的是，当将 b5 添加到体外重构系统中时，17OH-PregS 不会发生 17,20-碳-碳键断裂，而 b5 会促进 17OH-Preg 形成 DHEA。当使用表达 CYP17A1 和 CPR 的人 SOAT-HEK293 细胞时，我们可以确认 PregS 代谢为 17OH-PregS，从而强化了磺化类固醇途径的潜在生理意义。
In many tissues sulfonated steroids exceed the concentration of free steroids and recently they were also shown to fulfill important physiological functions. While it was previously demonstrated that cholesterol sulfate (CS) is converted by CYP11A1 to pregnenolone sulfate (PregS), further conversion of PregS has not been studied in detail. To investigate whether a steroidogenic pathway for sulfonated steroids exists similar to the one described for free steroids, we examined the interaction of PregS with CYP17A1 in a reconstitutedin-vitrosystem. Difference spectroscopy revealed aKd-value of 74.8 ± 4.2 μM for the CYP17A1–PregS complex, which is 2.5-fold higher compared to the CYP17A1–pregnenolone (Preg) complex. Mass spectrometry experiments proved for the first time that PregS is hydroxylated by CYP17A1 at position C17, identically to pregnenolone. A higherKm- and a lowerkcat-value for CYP17A1 using PregS compared with Preg were observed, indicating a 40% reduced catalytic efficiency when using the sulfonated steroid. Furthermore, we analyzed whether the presence of cytochromeb5(b5) has an influence on the CYP17A1 dependent conversion of PregS, as was demonstrated for Preg. Interestingly, with 17OH-PregS no scission of the 17,20-carbon–carbon bond occurs, whenb5is added to the reconstitutedin-vitrosystem, whileb5promotes the formation of DHEA from 17OH-Preg. When using human SOAT-HEK293 cells expressing CYP17A1 and CPR, we could confirm that PregS is metabolized to 17OH-PregS, strengthening the potential physiological meaning of a pathway for sulfonated steroids.