Endo Satoshi, Takada Sayaka, Honda Ryo P, Müller Kathrin, Weishaupt Jochen H, Andersen Peter M, Ludolph Albert C, Kamatari Yuji O, Matsunaga Toshiyuki, Kuwata Kazuo, El-Kabbani Ossama, Ikari Akira
Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196, Japan.
Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196, Japan.
Chem Biol Interact. 2017 Oct 1;276:194-202. doi: 10.1016/j.cbi.2016.12.018. Epub 2016 Dec 23.
Aldo-keto reductase (AKR) 1C3 is a cytosolic enzyme that metabolizes steroids, prostaglandins, toxic aldehydes and drugs. Recently, some nonsynonymous single nucleotide polymorphisms of AKR1C3 have been suggested to impact steroid and drug metabolism. In this study, we examined the effects of C154Y and L159V variants of AKR1C3 on stability and function of the enzyme. Both variants had been detected in patients with the neurodegenerative disease amyotrophic lateral sclerosis. Recombinant wild-type (WT), C154Y and L159V enzymes were similar in specific activity, but C154Y displayed much lower thermostability than WT and L159V. C154Y was inactivated by 10-min incubation at >25 °C, and about 90% of its activity was lost at 40 °C. Differential scanning fluorimetry revealed that T (thermal denaturation midpoint) of C154Y was lower than that of WT. In order to study the cause of thermosensitivity of C154Y, we prepared C154F and C154S mutant AKR1C3s. Like C154Y, C154F was highly sensitive to thermal inactivation, whereas C154S showed almost the same thermostability as WT. The C154F and C154Y variants induced secondary and tertiary structural changes in AKR1C3 at 40 °C as reflected by their altered circular dichroism and 8-anilinonaphthalene-1-sulfonate fluorescence characteristics. These results suggest that the replacement of C154 with a residue possessing a bulky aromatic side-chain impairs the folding of the α-helix containing C154 and its neighboring secondary structures, leading to low thermostability of AKR1C3. AKR1C3 metabolizes cytotoxic 4-oxo-2-nonenal into a less toxic metabolite, and overexpression of WT in HEK293 cells alleviated the 4-oxo-2-nonenal-induced cytotoxicity. In contrast, the overexpression of C154Y in the cells did not show such a significant protective effect, suggesting that C154Y is unstable in cells.
醛酮还原酶（AKR）1C3是一种胞质酶，可代谢类固醇、前列腺素、有毒醛类和药物。最近，有人提出AKR1C3的一些非同义单核苷酸多态性会影响类固醇和药物代谢。在本研究中，我们检测了AKR1C3的C154Y和L159V变体对该酶稳定性和功能的影响。这两种变体均在神经退行性疾病肌萎缩侧索硬化症患者中被检测到。重组野生型（WT）、C154Y和L159V酶的比活性相似，但C154Y的热稳定性远低于WT和L159V。在>25°C下孵育10分钟可使C154Y失活，在40°C时其活性丧失约90%。差示扫描荧光法显示C154Y的T（热变性中点）低于WT。为了研究C154Y热敏感性的原因，我们制备了C154F和C154S突变型AKR1C3。与C154Y一样，C154F对热失活高度敏感，而C154S的热稳定性几乎与WT相同。C154F和C154Y变体在40°C时诱导了AKR1C3的二级和三级结构变化，这通过它们改变的圆二色性和8-苯胺基萘-1-磺酸盐荧光特性得以体现。这些结果表明，用具有庞大芳香侧链的残基取代C154会损害包含C154的α-螺旋及其相邻二级结构的折叠，导致AKR1C3的热稳定性较低。AKR1C3将细胞毒性的4-氧代-2-壬烯醛代谢为毒性较小的代谢产物，在HEK293细胞中过表达WT可减轻4-氧代-2-壬烯醛诱导的细胞毒性。相比之下，在细胞中过表达C154Y并未显示出如此显著的保护作用，这表明C154Y在细胞中不稳定。
