Ezanno Hélène, le Bloc'h Jérôme, Beauchamp Erwan, Lagadic-Gossmann Dominique, Legrand Philippe, Rioux Vincent
Laboratoire de Biochimie-Nutrition Humaine, Agrocampus Ouest, INRA USC 2012, 65 rue de Saint-Brieuc, CS 84215, 35042, Rennes cedex, France.
Lipids. 2012 Feb;47(2):117-28. doi: 10.1007/s11745-011-3638-x. Epub 2011 Dec 3.
Dihydroceramide Δ4-desaturase 1 (DES1) catalyzes the last step of the de novo ceramide biosynthesis, which consists of the introduction of a trans Δ4-double bond in the carbon chain of the dihydroceramide. It was previously observed that myristic acid binds DES1 through N-myristoylation. This N-terminal modification significantly increased the activity of the recombinant DES1 in COS-7 cells and targeted part of the enzyme initially present in the endoplasmic reticulum to the mitochondrial outer membrane, leading to an increase in ceramide levels. Since these results were obtained in a recombinant COS-7 cell model with high expression of rat DES1, the purpose of the present study was to investigate if the native DES1 enzyme was really upregulated by its N-myristoylation in cultured rat hepatocytes. We first showed that DES1 was the main dihydroceramide desaturase isoform expressed in rat hepatocytes. In this model, the wild-type myristoylable recombinant form of rat DES1 was found in both the endoplasmic reticulum and the mitochondria whereas the mutated non-myristoylable recombinant form (N-terminal glycine replaced by an alanine) was almost exclusively localized in the endoplasmic reticulum, which evidenced the importance of the myristoylation. Then, we showed that compared to other fatty acids, myristic acid was the only one to increase native DES1 activity, in both total cell lysates and mitochondrial fractions. The myristic acid-associated increase in DES1 activity was not linked to elevated mRNA or protein expression but more likely to its N-terminal myristoylation. Finally, the myristic acid-associated increase in DES1 activity slightly enhanced the number of apoptotic cells.
二氢神经酰胺Δ4-去饱和酶1（DES1）催化神经酰胺从头生物合成的最后一步，该步骤包括在二氢神经酰胺的碳链中引入反式Δ4-双键。此前观察到肉豆蔻酸通过N-肉豆蔻酰化与DES1结合。这种N端修饰显著提高了重组DES1在COS-7细胞中的活性，并将最初存在于内质网中的部分酶靶向线粒体外膜，导致神经酰胺水平升高。由于这些结果是在高表达大鼠DES1的重组COS-7细胞模型中获得的，本研究的目的是调查天然DES1酶在培养的大鼠肝细胞中是否真的通过其N-肉豆蔻酰化而上调。我们首先表明DES1是大鼠肝细胞中表达的主要二氢神经酰胺去饱和酶同工型。在该模型中，内质网和线粒体中均发现了大鼠DES1的野生型可肉豆蔻酰化重组形式，而突变的不可肉豆蔻酰化重组形式（N端甘氨酸被丙氨酸取代）几乎完全定位于内质网，这证明了肉豆蔻酰化的重要性。然后，我们表明，与其他脂肪酸相比，肉豆蔻酸是唯一能增加天然DES1活性的脂肪酸，无论是在总细胞裂解物还是线粒体组分中。肉豆蔻酸相关的DES1活性增加与mRNA或蛋白质表达升高无关，而更可能与其N端肉豆蔻酰化有关。最后，肉豆蔻酸相关的DES1活性增加略微增加了凋亡细胞的数量。
