Research paperN-Myristoylation targets dihydroceramide Δ4-desaturase 1 to mitochondria: Partial involvement in the apoptotic effect of myristic acid
Introduction
Since adipocytes only have the ability to store fatty acids (FA) in excess by accumulating triglycerides, lipid overload in nonadipose cells leads to major metabolic disorders. High concentrations of free FA incubated with ventricular myocytes [1], Chinese Hamster Ovary cells [2], pancreatic β-cells [3], breast cancer cells [4] and also hepatocytes [5] induce apoptosis, a mechanism that has therefore been called lipoapoptosis [6]. All these studies have suggested that this phenomenon is specific of saturated free FA, especially palmitic acid (C16:0) and stearic acid (C18:0). In vivo, high levels of dietary saturated FA also reduced the proliferative capacity of liver cells and promoted liver injury [7]. Two complementary type of mediation have been explored to explain this type of lipotoxicity, involving or not ceramide. First, palmitic and stearic acids are both able to induce partial mitochondria uncoupling [8], endoplasmic reticulum stress [9] and to generate reactive oxygen species (ROS) [2] and nitric oxide [10]. Second, apoptosis induced by these saturated free FA is correlated with de novo synthesis of ceramide [11].
Ceramide consists of a sphingosine backbone produced by the condensation of serine with palmitoyl-CoA, to which a variety of FA are attached via an amide bond. It can be formed either from sphingomyelin hydrolysis by distinct sphingomyelinases or from de novo synthesis [12]. This basic structure has been demonstrated to be a bioactive molecule involved in stress response [13], functional rafts [14], [15] and also in apoptosis [16]. In ceramide-mediated apoptosis, mitochondria have been identified as the preferential target. Indeed, ceramide is able to interact directly with the mitochondrial electron transport chain [17], to induce ROS production [18], to form channels in the mitochondrial outer membranes [19], [20] and finally to mediate the release of key apoptosis-inducing proteins into the cytoplasm [21].
Interestingly, ceramide, but not its precursor dihydroceramide, is able to induce these events [17], [18], [21]. In this context, the last step of the ceramide de novo synthesis pathway, i.e. the introduction of a trans Δ4,5-double bond into the dihydroceramide carbon chain, catalyzed by the dihydroceramide Δ4-desaturase (DES), seems to be critical for the acquisition of the bioactive properties of ceramide. Recently, two different isoforms of dihydroceramide Δ4-desaturases (DES1 and DES2) have been identified in mouse, rat and human [22], [23], [24]. The regulation of these enzymes is still poorly understood, but it was suggested that DES isoforms are part of a cytochrome b5 electron transport system, like FA desaturases [25]. More recently, we have demonstrated that both rat DES1 and DES2 are myristoylated in vivo [23]. Myristoylation corresponds to the linkage between the N-terminal glycine residue of the protein and myristic acid (C14:0) via an amide bond [26]. This N-terminal modification significantly increased the activity of recombinant DES1 [23].
The description of this regulatory mechanism highlighted a new potential relationship between myristic acid, the saturated FA capable of binding and activating the enzyme involved in the final de novo ceramide biosynthesis step, and lipoapoptosis induced through the ceramide pathway. Therefore, in this study we hypothesized and showed that the myristoylation of dihydroceramide Δ4-desaturase 1 could target part of the enzyme to the mitochondria, leading to an increase in ceramide levels (specifically in the mitochondria) which in turn leads to apoptosis of COS-7 cells. Since myristic acid alone also possesses an additional pro-apoptotic effect, this newly described mechanism contributes to partially explain the apoptosis effect induced by myristic acid.
Section snippets
Chemicals
(1-14C)-octanoic acid was purchased from American Radiolabeled Chemicals (Isobio, Fleurus, Belgium). (1-14C)-N-octanoyldihydroceramide was chemically synthesized in our laboratory, as previously described [23], [27]. Reagents for electrophoretical application were from GE Healthcare (Orsay, France) and BioRad (Marnes-la-Coquette, France). Solvents (HPLC grade) came from Fisher Scientific (Elancourt, France). Other chemicals were obtained from VWR (Fontenay-sous-Bois, France) or from Sigma
The myristoylated form of DES1 is localized not only in the endoplasmic reticulum but also in mitochondria in COS-7 cells incubated with myristic acid
We have previously shown that dihydroceramide Δ4-desaturase 1 activity is regulated by myristic acid through its N-terminal myristoylation [23]. In the present work, we hypothesized that DES1 myristoylation could also modify its subcellular localization. We first compared the intracellular distribution of the recombinant wild-type (DES1-Gly) and mutant unmyristoylable (DES1-Ala) forms of dihydroceramide Δ4-desaturase 1, by using an immunofluorescence assay on transiently transfected COS-7 cells
Discussion
Saturated FA can increase ceramide concentration and induce apoptosis [11]. Surprisingly, all the studies claiming these results have been designed by using only palmitic and stearic acids as the saturated FA. In terms of co- and post-translational protein modification, myristic acid is, for instance, the single saturated FA capable of myristoylating proteins [26]. Protein N-myristoylation can modify the conformation of the protein, change protein–protein or protein–membrane interactions. In
Acknowledgements
The authors are indebted to ARILAIT RECHERCHES (France) for constructive scientific discussion and financial support. The antibody to PEMT2 was a generous gift from Dr. D.E. Vance (University of Alberta, Edmonton, Canada). We thank N. Monthéan, and R. Marion for able technical assistance and animal care. We thank Dr. G. Jan (STLO, INRA, Rennes) for the use of the epifluorescence microscope.
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