Gupta Sita D, Gable Kenneth, Alexaki Aikaterini, Chandris Panagiotis, Proia Richard L, Dunn Teresa M, Harmon Jeffrey M
From the Departments of Biochemistry and Molecular Biology and.
Genetics of Development and Disease Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 2015 Jan 2;290(1):90-8. doi: 10.1074/jbc.M114.588236. Epub 2014 Nov 13.
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.
在哺乳动物HEK293细胞中研究了丝氨酸棕榈酰转移酶(SPT)活性与鞘脂生物合成的ORMDL调节之间的关系。三种人类ORMDL中的每一种都降低了野生型SPT或在hLCB1中含有C133W突变的SPT(产生不可分解代谢的鞘氨醇碱1-脱氧鞘氨醇)过表达后长链碱基合成的增加。无论SPT是作为单个亚基还是作为异源三聚体单链SPT融合蛋白表达,鞘氨醇碱合成的ORMDL依赖性抑制都会发生。在稳定转染的细胞中,在四环素诱导型启动子的控制下过表达单链SPT融合蛋白导致内源性ORMDL表达增加。这种增加不是转录性的;任何一种ORMDL mRNA均无显著增加。增加的ORMDL蛋白表达需要SPT活性,因为在hLCB2a中具有突变的催化无活性的SPT过表达几乎没有影响。重要的是,myriocin对SPT的抑制以及伏马菌素对神经酰胺合成酶的抑制也阻断了ORMDL表达的增加,这表明表达增加是对代谢信号的一种反应。此外,用伏马菌素处理阻断ORMDL诱导导致体内SPT活性的增加明显大于允许ORMDL增加时观察到的增加,证明了这种反应的生理意义。