Abe Fumiyoshi, Usui Keiko, Hiraki Toshiki
Molecular Evolution and Adaptation Research, Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka 237-0061, Japan.
Biochemistry. 2009 Sep 15;48(36):8494-504. doi: 10.1021/bi900578y.
Azole anitifungal drugs such as fluconazole inhibit 14alpha-demethylase. The mechanism of fluconazole action on the plasma membrane is assumed to be ergosterol depletion and accumulation of a toxic sterol, 14alpha-methyl-3,6-diol, that differs in C-6 hydroxylation, B-ring saturation, C-14 methylation, and side-chain modification. Nevertheless, little is known about how these sterol modifications mechanically influence membrane properties and hence fungal viability. Employing time-resolved measurement with a fluorescence anisotropy probe, 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), we demonstrated that fluconazole administration decreased the rigidity of the plasma membrane of Saccharomyces cerevisiae, leading to a dramatic reduction in the order parameter (S) from 0.965 to 0.907 and a 5-fold acceleration of the rotational lipid motion. This suggests that the altered sterol has a deleterious impact on membrane packing, resulting in increased fluidity. Deletion of ERG3 confers hyperresistance to fluconazole by circumventing the accumulation of 14alpha-methyl-3,6-diol and instead produces 14alpha-methylfecosterol lacking the 6-OH group. We found that ERG3 deletion mitigated the fluconazole-induced loss of membrane rigidity with S remaining at a higher value (=0.922), which could contribute to the fluconazole resistance in the erg3Delta mutant. The reduced ability of the 6-OH sterol to stiffen lipid bilayers was supported by the finding that 30 mol % of 6alpha-hydroxy-5alpha-cholestanol marginally increased the S value of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes, while cholesterol and dihydrocholesterol markedly increased it. The decay of the TMA-DPH fluorescence was bimodal in the wild-type strain. This heterogeneity could have arisen from varying degrees of water penetration into the plasma membrane. Fluconazole eliminated the heterogeneity of the dielectric characteristic of the membrane interfacial region, and concomitantly the TMA-DPH lifetime was shortened. Therefore, we conclude that 14alpha-methyl-3,6-diol is insufficient to pack the plasma membrane, allowing water penetration, which is consistent with membrane disorder after fluconazole administration. Our findings illustrate the role of ergosterol in maintaining membrane heterogeneity and preventing water penetration as well as maintaining the rigidity of the plasma membrane interfacial region.
唑类抗真菌药物如氟康唑可抑制14α-去甲基化酶。氟康唑作用于质膜的机制被认为是麦角甾醇耗竭以及一种有毒甾醇14α-甲基-3,6-二醇的积累，该甾醇在C-6羟基化、B环饱和度、C-14甲基化和侧链修饰方面存在差异。然而，关于这些甾醇修饰如何在机械上影响膜特性进而影响真菌活力，人们知之甚少。我们使用荧光各向异性探针1-[4-(三甲基氨基)苯基]-6-苯基-1,3,5-己三烯（TMA-DPH）进行时间分辨测量，结果表明给予氟康唑会降低酿酒酵母质膜的刚性，导致序参数（S）从0.965显著降至0.907，并且脂质旋转运动加速了5倍。这表明改变后的甾醇对膜的堆积产生了有害影响，导致流动性增加。ERG3基因的缺失通过避免14α-甲基-3,6-二醇的积累赋予对氟康唑的超抗性，并转而产生缺乏6-OH基团的14α-甲基粪甾醇。我们发现ERG3基因缺失减轻了氟康唑诱导的膜刚性丧失，S值保持在较高水平（=0.922），这可能有助于erg3Δ突变体对氟康唑产生抗性。6-OH甾醇使脂质双层变硬的能力降低，这一发现得到了支持，即30 mol%的6α-羟基-5α-胆甾烷醇仅略微增加了1-棕榈酰-2-油酰-sn-甘油-3-磷酸胆碱膜的S值，而胆固醇和二氢胆固醇则显著增加了该值。在野生型菌株中，TMA-DPH荧光的衰减是双峰的。这种异质性可能是由于水渗透到质膜的程度不同所致。氟康唑消除了膜界面区域介电特性的异质性，同时TMA-DPH的寿命缩短。因此，我们得出结论，14α-甲基-3,6-二醇不足以填充质膜，从而允许水渗透，这与给予氟康唑后膜的无序状态一致。我们的研究结果说明了麦角甾醇在维持膜的异质性、防止水渗透以及维持质膜界面区域刚性方面的作用。
