Expression of the ORMDLS, Modulators of Serine Palmitoyltransferase, Is Regulated by Sphingolipids in Mammalian Cells
Expression of the ORMDLS, Modulators of Serine Palmitoyltransferase, Is Regulated by Sphingolipids in Mammalian Cells
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DOI：
10.1074/jbc.m114.588236
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发表时间：
2015-01-02
影响因子：
4.8
通讯作者：
Harmon, Jeffrey M.
中科院分区：
文献类型：
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作者：
Gupta, Sita D.;Gable, Kenneth;Harmon, Jeffrey M.
背景：与它们的酵母直向同源物相反，哺乳动物ORMDLs调节丝氨酸棕榈酰转移酶的机制尚不清楚。结果如下：HEK 293细胞中丝氨酸棕榈酰转移酶的过表达导致长链碱基合成增加和ORMDL表达的活性依赖性增加。结论：神经酰胺合成酶的一个产物介导ORMDL表达的调节和丝氨酸棕榈酰转移酶的抑制。重要性：丝氨酸棕榈酰转移酶（serine palmitoyltransferase，SPT）活性间接调控ORMDL的表达。三种人ORMDLs中的每一种都减少了在野生型SPT或含有hLCB 1中C133 W突变的SPT过表达后观察到的长链碱基合成的增加，所述突变产生不可分解代谢的鞘氨醇类碱基1-脱氧Sa。无论SPT表达为单个亚基还是异源三聚体单链SPT融合蛋白，都发生了类鞘氨醇碱合成的ORMDL依赖性抑制。在稳定转染的细胞中，四环素诱导型启动子控制下的单链SPT融合蛋白的过表达导致内源性ORMDL表达增加。这种增加不是转录的;任何ORMDL mRNA都没有显著增加。增加ORMDL蛋白表达需要SPT活性，因为在hLCB 2a中突变的催化失活SPT的过表达几乎没有影响。值得注意的是，增加的ORMDL表达也被SPT的多球菌素抑制以及神经酰胺脱氢酶的伏马菌素抑制所阻断，这表明增加的表达是对代谢信号的响应。此外，用伏马菌素处理阻断ORMDL诱导导致体内SPT活性比允许ORMDL增加时所见的显著更大的增加，证明了这种应答的生理意义。
Background: In contrast to their yeast orthologues, the mechanism by which mammalian ORMDLs regulate serine palmitoyltransferase is not understood. Results: Overexpression of serine palmitoyltransferase in HEK293 cells results in increased long-chain base synthesis and an activity-dependent increase in ORMDL expression. Conclusion: A product of ceramide synthase mediates regulation of ORMDL expression and inhibition of serine palmitoyltransferase. Significance: Serine palmitoyltransferase activity indirectly regulates ORMDL expression.The relationship between serine palmitoyltransferase (SPT) activity and ORMDL regulation of sphingolipid biosynthesis was investigated in mammalian HEK293 cells. Each of the three human ORMDLs reduced the increase in long-chain base synthesis seen after overexpression of wild-type SPT or SPT containing the C133W mutation in hLCB1, which produces the non-catabolizable sphingoid base, 1-deoxySa. ORMDL-dependent repression of sphingoid base synthesis occurred whether SPT was expressed as individual subunits or as a heterotrimeric single-chain SPT fusion protein. Overexpression of the single-chain SPT fusion protein under the control of a tetracycline-inducible promoter in stably transfected cells resulted in increased endogenous ORMDL expression. This increase was not transcriptional; there was no significant increase in any of the ORMDL mRNAs. Increased ORMDL protein expression required SPT activity since overexpression of a catalytically inactive SPT with a mutation in hLCB2a had little effect. Significantly, increased ORMDL expression was also blocked by myriocin inhibition of SPT as well as fumonisin inhibition of the ceramide synthases, suggesting that increased expression is a response to a metabolic signal. Moreover, blocking ORMDL induction with fumonisin treatment resulted in significantly greater increases in in vivo SPT activity than was seen when ORMDLs were allowed to increase, demonstrating the physiological significance of this response.