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  • Third DGK protein stabilization by myristic acid is cell lin


    Third, DGKδ2 protein stabilization by myristic Cilengitide receptor is cell line specific. Myristic acid had no obvious effects on DGKδ protein stability in the liver cell line HepG2, pancreas cell line MIA-PaCa-2 and neuronal cell line Neuro-2a cells (Fig. 5). Although myristic acid moderately increased the DGKδ2 protein level (46% increase) in kidney-derived HEK293 cells, the fatty acid augmented the protein level more strongly (100% increase) in C2C12 myotube cells (Fig. 5). Speziali et al. recently reported that the protein levels of only 32 proteins were specifically increased by myristic acid in HepG2 cells based on proteomics analysis [28], which indicates that myristic acid regulates the levels of a limited number of proteins. Moreover, in line with our results, the DGKδ2 protein level was not changed in HepG2 cells. This report supports our conclusion that the myristic acid-dependent regulation of protein levels is protein- and cell line-dependent. However, it is unclear whether the effects of myristic acid on protein levels in HepG2 cells were the result of protein stabilization or transcriptional regulation. The results shown in Fig. 6 suggest that the ubiquitin-proteasome system and the autophagy-lysosome pathway participate, at least in part, in DGKδ2 degradation. However, even the use of a combination of high concentrations of a proteasome inhibitor, MG132, and a lysosome inhibitor, chloroquine, did not fully recover the DGKδ2 levels during protein degradation (Fig. 6), which suggests that the other pathway(s) are involved in the DGKδ2 degradation. Therefore, it is possible that DGKδ2 is degraded in a complex manner. How does myristic acid regulate the stability of DGKδ2 protein? There is a possibility that peroxisome proliferator-activated receptors, fatty acid-binding proteins and/or free fatty acid receptors, which bind to fatty acids, mediate the regulation of protein stabilization. However, this possibility is not likely because peroxisome proliferator-activated receptors [29,30], fatty acid-binding proteins [31,32] and free fatty acid receptors [33,34] do not possess high myristic acid-selectivity. In contrast, the high specificity of myristic acid in its effect on DGKδ2 protein stabilization implies that myristoylation regulates this event. Given that myristoylation is involved in DGKδ2 protein stabilization, it is likely that myristoylation indirectly affects the stabilization because DGKδ2 [35] contains no consensus sequences that would serve as targets of myristoylation [8]. Several reports have demonstrated that N-myristoylation is involved in the control of the ubiquitin-proteasome system. For example, the activity of the proteasome is diminished by the N-myristoylation of SWOF1 in Aspergillus nidulans [36]. N-myristoylation of an E3 ubiquitin ligase, Neuralized-like 1, affects its ability to down-regulate Jagged1 [37]. In addition to the ubiquitin-proteasome system, myristoylation was also reported to regulate autophagy of the huntingtin protein [38]. However, in this case, myristoylation enhanced the autophagy (destabilization) of huntingtin [38]. We previously reported that the changes in the expression level of DGKδ2 affect C2C12 myogenic differentiation [39]. Proteostasis requires both protein synthesis and degradation. Indeed, protein degradation via the ubiquitin-proteasome and autophagy-lysosome systems is important for skeletal muscle maintenance [40]. Thus, myristic acid may be able to regulate skeletal muscle development and myogenic differentiation via stabilization of the DGKδ2 protein. Chronic administration of myristic acid was shown to increase the levels of DGKδ2 protein and improve hyperglycaemia in Nagoya-Shibata-Yasuda congenital type 2 diabetic mice [22]. Therefore, it is possible that myristic acid can reduce the risk of type 2 diabetes in human. In the present study, we demonstrated that myristic acid stabilizes DGKδ2 protein in a highly selective manner. Thus, the administration of myristic acid is anticipated to have few unexpected side effects.