Sphingolipid synthesis as a target for antifungal drugs - Complementation of the inositol phosphorylceramide synthase defect in strain of Saccharomyces cerevisiae by the AUR1 gene
Sphingolipid synthesis as a target for antifungal drugs - Complementation of the inositol phosphorylceramide synthase defect in strain of Saccharomyces cerevisiae by the AUR1 gene
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DOI：
10.1074/jbc.272.15.9809
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发表时间：
1997-04-11
影响因子：
4.8
通讯作者：
Dickson, RC
中科院分区：
文献类型：
--
作者：
Nagiec, MM;Nagiec, EE;Dickson, RC
我们已经鉴定了一个酿酒酵母基因，该基因在神经酰胺中加入肌醇磷酸生成肌醇-β-神经酰胺，该反应由磷脂酰肌醇：神经酰胺磷酸肌醇转移酶(IPC合成酶)催化。这一步应该是抗真菌药物的有效靶点。我们实验中的一个关键因素是开发了一种程序，用于分离在第一步神经鞘脂脂合成下游步骤中存在缺陷的突变株，包括IPC合成酶中存在缺陷的突变株。数据显示，突变菌株未能将放射性肌醇或N-乙酰鞘氨酸掺入鞘磷脂中，并通过使用一种改进的检测方法，证明突变菌株缺乏酶活性，从而支持IPC合酶缺陷。此外，对于缺乏IPC合成酶活性的菌株来说，当给予外源植鞘糖苷时，突变体积累了神经酰胺。神经酰胺的积累伴随着细胞的死亡，这表明酵母中存在神经酰胺激活的死亡反应。分离到一个补充IPC合酶缺陷、恢复酶活性和鞘脂合成的基因AUR1(YKL004w)。AUR1的突变已经被证明对抗真菌药物Areobasidin A具有耐药性，这使得我们预测该药物应该抑制IPC合成酶活性。我们的数据显示，该药物是一种有效的IPC合成酶抑制剂，其IC50约为0.2 nM。真菌病原体对人类健康的威胁越来越大。既然IPC合成酶已经被证明是金黄色葡萄糖素A的靶标，那么就有可能开发高通量的筛选来寻找新的IPC合成酶抑制剂来对抗真菌疾病。
We have identified a Saccharomyces cerevisiae gene necessary for the step in sphingolipid synthesis in which inositol phosphate is added to ceramide to form inositol-P-ceramide a reaction catalyzed by phosphatidylinositol:ceramide phosphoinositol transferase (IPC synthase). This step should be an effective target for antifungal drugs. A key element in our experiments was the development of a procedure for isolating mutants defective in steps in sphingolipid synthesis downstream from the first step including a mutant defective in IPC synthase. An IPC synthase defect is supported by data showing a failure of the mutant strain to incorporate radioactive inositol or N-acetylsphinganine into sphingolipids and, by using an improved assay, a demonstration that the mutant strain lacks enzyme activity. Furthermore, the mutant accumulates ceramide when fed exogenous phytosphingosine as expected for a strain lacking IPC synthase activity. Ceramide accumulation is accompanied by cell death, suggesting the presence of a ceramide-activated death response in yeast. A gene, AUR1 (YKL004w), that complements the IPC synthase defect and restores enzyme activity and sphingolipid synthesis was isolated. Mutations in AUR1 had been shown previously to give resistance to the antifungal drug aureobasidin A, leading us to predict that the drug should inhibit IPC synthase activity. Our data show that the drug is a potent inhibitor of IPC synthase with an IC50 of about 0.2 nM. Fungal pathogens are an increasing threat to human health. Now that IPC synthase has been shown to be the target for aureobasidin A, it should be possible to develop high throughput screens to identify new inhibitors of IPC synthase to combat fungal diseases.