Straus Alexandra J, Mavodza Grace, Senkal Can E
Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, VA, USA; C. Kenneth and Dianne Wright Center for Clinical and Translational Research, Virginia Commonwealth University School of Medicine, Richmond, VA, USA.
Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, VA, USA.
J Lipid Res. 2025 Jan;66(1):100715. doi: 10.1016/j.jlr.2024.100715. Epub 2024 Nov 26.
Sphingolipids play key roles in membrane structure and cellular signaling. Ceramide synthase (CerS)-generated ceramide is implicated in cellular stress responses and induction of apoptosis. Ceramide and other sphingolipids are linked to the induction of ER stress response pathways. However, the mechanisms by which ceramide modulates ER stress signaling are not well understood. Here, we show that the ER stress inducer brefeldin A (BFA) causes increased glycosylation of CerS6, and that treatment with BFA causes increased endogenous ceramide accumulation. To our surprise, we found that CerS6 activity was not affected by BFA-induced glycosylation. Instead, our data show that basal glycosylation of CerS6 at Asn18 is required for CerS6 activity. We used a robust HCT116 CRISPR-Cas9 CerS6 KO with reintroduction of either WT CerS6 or a mutant CerS6 with a point mutation at asparagine-18 to an alanine (N18A) which abrogated glycosylation at that residue. Our data show that cells stably expressing the N18A mutant CerS6 had significantly lower activity in vitro and in situ as compared to WT CerS6 expressing cells. Further, the defective CerS6 with N18A mutation also had defects in GSK3β, AKT, JNK, and STAT3 signaling. Despite being required for CerS6 activity, Asn18 glycosylation did not influence ER stress response pathways. Overall, our study provides vital insight into the regulation of CerS6 activity by posttranslational modification at Asn18 and identifies glycosylation of CerS6 to be important for ceramide generation and regulation of downstream cellular signaling pathways.
鞘脂在膜结构和细胞信号传导中起关键作用。神经酰胺合酶（CerS）产生的神经酰胺与细胞应激反应和细胞凋亡的诱导有关。神经酰胺和其他鞘脂与内质网应激反应途径的诱导有关。然而，神经酰胺调节内质网应激信号传导的机制尚不清楚。在这里，我们表明内质网应激诱导剂布雷菲德菌素A（BFA）会导致CerS6的糖基化增加，并且用BFA处理会导致内源性神经酰胺积累增加。令我们惊讶的是，我们发现CerS6的活性不受BFA诱导的糖基化影响。相反，我们的数据表明，CerS6在Asn18处的基础糖基化是CerS6活性所必需的。我们使用了一种强大的HCT116 CRISPR-Cas9 CerS6基因敲除细胞系，重新引入野生型CerS6或在天冬酰胺-18处有一个点突变（N18A）的突变型CerS6，该突变消除了该残基处的糖基化。我们的数据表明，与表达野生型CerS6的细胞相比，稳定表达N18A突变型CerS6的细胞在体外和原位的活性显著降低。此外，具有N18A突变的缺陷型CerS6在GSK3β、AKT、JNK和STAT3信号传导方面也存在缺陷。尽管Asn18糖基化是CerS6活性所必需的，但它并不影响内质网应激反应途径。总体而言，我们的研究为通过Asn18的翻译后修饰对CerS6活性的调节提供了重要见解，并确定CerS6的糖基化对神经酰胺生成和下游细胞信号传导途径的调节很重要。
