Characterization of the Candida glabrata Transcription Factor CgMar1: Role in Azole Susceptibility

The prevalence of antifungal resistance in Candida glabrata, especially against azole drugs, results in difficult-to-treat and potentially life-threatening infections. Understanding the molecular basis of azole resistance in C. glabrata is crucial to designing more suitable therapeutic strategies. In this study, the role of the transcription factor encoded by ORF CAGL0B03421g, here denominated as CgMar1 (Multiple Azole Resistance 1), in azole susceptibility was explored. Using RNA-sequencing, CgMar1 was found to regulate 337 genes under fluconazole stress, including several related to lipid biosynthesis pathways. In this context, CgMar1 and its target CgRSB1, encoding a predicted sphingoid long-chain base efflux transporter, were found to contribute to plasma membrane sphingolipid incorporation and membrane permeability, decreasing fluconazole accumulation. CgMar1 was found to associate with the promoter of CgRSB1, which contains two instances of the CCCCTCC consensus, found to be required for CgRSB1 activation during fluconazole stress. Altogether, a regulatory pathway modulating azole susceptibility in C. glabrata is proposed, resulting from what appears to be a neofunctionalization of a Hap1-like transcription factor.

Impaired skin barrier function due to reduced ω- O -acylceramide levels in a mouse model of Sjögren–Larsson syndrome

Sjögren–Larsson syndrome (SLS) is an inherited neurocutaneous disorder whose causative gene encodes the fatty aldehyde dehydrogenase ALDH3A2. To date, the detailed molecular mechanism of the skin pathology of SLS has remained largely unclear. We generated double knockout (DKO) mice for Aldh3a2 and its homolog Aldh3b2 (a pseudogene in humans). These mice showed hyperkeratosis and reduced fatty aldehyde dehydrogenase activity and skin barrier function. The levels of ω- O -acylceramides (acylceramides), which are specialized ceramides essential for skin barrier function, in the epidermis of DKO mice were about 60% of those in wild type mice. In the DKO mice, levels of acylceramide precursors (ω-hydroxy ceramides and triglycerides) were increased, suggesting that the final step of acylceramide production was inhibited. A decrease in acylceramide levels was also observed in human immortalized keratinocytes lacking ALDH3A2 . Differentiated keratinocytes prepared from the DKO mice exhibited impaired long-chain base metabolism. Based on these results, we propose that the long-chain-base–derived fatty aldehydes that accumulate in DKO mice and SLS patients attack and inhibit the enzyme involved in the final step of acylceramide. Our findings provide insight into the pathogenesis of the skin symptoms of SLS, i.e., decreased acylceramide production, and its molecular mechanism.

Triptolide Inhibits the Biological Processes of HUVEC and HepG2 Cells via the Serine Palmitoyltransferase Long Chain Base Subunit 2/sphingosine-1-phosphate Signaling Pathway

Background: Hepatocellular carcinoma is a cancer that has a high incidence in men, and its incidence is increasing year by year. Studies show that angiogenesis plays an important role in the formation of tumors, not only providing nutrients to tumor cells, but also closely related to tumor growth and metastasis. So, how to find new anti-vascular and anti-tumor targets for the pathogenesis of liver cancer is a key issue that needs to be resolved.Methods: After treating Human umbilical vein endothelial cells (HUVEC) and HepG2 cells with different concentrations of triptolide (TP), the relationship between TP's anti-vascular and anti-tumor activities and sphingolipids was investigated respectively. Then, the three-dimensional co-culture model was used to explore the correlation between HUVEC and HepG2 cells, and to find the relationship between it and sphingolipids.Results: This study explored the effects of TP on the proliferation, migration, adhesion and tube formation of HUVEC cells, as well as the effects on the proliferation, migration and invasion of HepG2 cells. And through the PCR Array assay to screen the changes of related sphingolipid genes, it was found that serine palmitoyltransferase long chain base subunit 2 (SPTLC2) was most likely to be related to the effect of TP. In further transfection experiments, it was found that down-regulation of SPTLC2 can inhibit the proliferation, migration, adhesion and tube formation of HUVEC cells, and down-regulation of SPTLC2 can also inhibit the proliferation, migration and invasion of HepG2 cells. The up-regulation of SPTLC2 has opposite effects in these two cell lines. In the three-dimensional co-culture model of HUVEC and HepG2 cells, it was found for the first time that HUVEC cells can promote the biological process of HepG2 cells. It was found through enzyme linked immunosorbent assay (ELISA) and western blot experiments that it may be achieved through the sphingosine-1-phosphate (S1P)/S1P receptors (S1PRs) pathway. Finally, we found that the promotion effect of HUVEC cells on HepG2 cells was significantly inhibited after HUVEC cells were treated with TP.Conclusions: These data confirmed that the level of SPTLC2 may be related to the anti-vascular and anti-tumor effects of TP. The data also showed that there was a correlation between the viability of HepG2 cells and HUVEC cells, which may be related to the expression of S1P/S1PRS. Ultimately, these data may help discover new anti-tumor targets.

Direct LC–ESI–MS/MS analysis of plant glucosylceramide and ceramide species with 8E and 8Z isomers of the long chain base

ABSTRACT Glucosylceramides and ceramides with 8E and 8Z isomers of the long chain base are found in plants. These isomers have been difficult to quantify separately using liquid chromatography–tandem mass spectrometry (LC–MS/MS) because the isomers have the same retention time, their precursor and product ions have the same m/z values, and plant ceramide standards are not commercially available. Here we tested trial separations using various ODS columns and prepared plant ceramide standards generated by human glucocerebrosidase (imiglucerase) using commercially available plant glucosylceramide standards as the substrates. Consequently, we were able to quantify the isomers based on differences in retention times in a TSKgel ODS-120A column (Tosoh, Tokyo Japan) using LC–electrospray ionization–MS/MS (LC–ESI–MS/MS).

Phosphorylation of LCB1 subunit of serine palmitoyltransferase stimulates its activity and modulates sphingolipid biosynthesis in Arabidopsis

Sphingolipids are the structural elements for membrane lipid bilayers and the signal molecules for many cellular processes. Serine palmitoyltransferase (SPT) is the first committed and rate-limiting enzyme in the de novo sphingolipids biosynthetic pathway. The core SPT was previously suggested as a heterodimer consisting of LCB1 and LCB2 subunits. The SPT activity was shown to be inhibited by orosomucoid proteins (ORMs) and stimulated by small subunits of SPT (ssSPT). However, whether LCB1 is modified and how the modification regulates SPT activity have been unclear. Here, we show that activation of MPK3 and MPK6 by upstream MKK9 and Flg22 (a pathogen-associated molecular pattern) treatment increases SPT activity and induces the accumulation of sphingosine long-chain base (LCB) t18:0 in Arabidopsis thaliana; the activated MPK3 and MPK6 phosphorylate AtLCB1. Phosphorylation of AtLCB1 strengthens its binding with AtLCB2b, promotes its binding with ssSPTs, and stimulates the higher-order-oligomer and active SPT complexes formation. Our findings suggest a novel regulatory mechanism of SPT activity.

Long-Chain Base (LCB)-Targeted Lipidomics Study Uncovering the Presence of a Variety of LCBs in Mammalian Blood

Globotriaosylsphingosine (LysoGb3) is a biomarker for Fabry disease (OMIM 301500) that contains long-chain bases (LCBs) as a building block. There have been several studies proposing that LysoGb3 forms with distinct LCBs could be putative disease subtype-related biomarkers for this congenital disorder; however, there have been no detailed multiple reaction monitoring-based studies examining the LCB distribution in this lysosphingolipid. To achieve this, we established an assay procedure that aimed at elucidating the LCB-targeted lipidome using liquid chromatography–tandem mass spectrometry. Consistent with previous studies, we found d18:1 to be the major LCB species of the LysoGb3 in pooled human plasma, while some atypical LCBs, such as d18:2, d18:0, t18:1, d16:1, and d17:1, were detected as minor fractions. When the same methodology was applied to fetal bovine serum (FBS) as a positive control, we identified additional unique LCB species, such as t18:0, d20:1, t19:1, and t21:1, in herbivore LysoGb3. Furthermore, we found an elevation of sphingosine and LysoGb3, which are N-deacylated forms of ceramide and Gb3, respectively, in FBS, suggesting that ceramidase activity may be involved in this process. Thus, our LCB-targeted lipidomics data revealed that mammalian LCBs in glycosphingolipids have a greater variety of molecular species than previously expected.