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The UDP-glucose ceramide glycosyltransferase (UGCG) is a key enzyme in the sphingolipid metabolism by generating glucosylceramide (GlcCer), the precursor for all glycosphingolipids (GSL), which are essential for proper cell function. Interestingly, the UGCG is also overexpressed in several cancer types and correlates with multidrug resistance protein 1 ( MDR1 ) gene expression. This membrane protein is responsible for efflux of toxic substances and protects cancer cells from cell damage through chemotherapeutic agents. Studies showed a connection between UGCG and MDR1 overexpression and multidrug resistance development, but the precise underlying mechanisms are unknown. Here, we give an overview about the UGCG and its connection to MDR1 in multidrug resistant cells. Furthermore, we focus on UGCG transcriptional regulation, the impact of UGCG on cellular signaling pathways and the effect of UGCG and MDR1 on the lipid composition of membranes and how this could influence multidrug resistance development. To our knowledge, this is the first review presenting an overview about UGCG with focus on the relationship to MDR1 in the process of multidrug resistance development.

Glucosylceramide (GlcCer), a common precursor of different glycosphingolipids, is shown to be channelled to two distinct pathways in the Golgi; non-vesicular transport from the cis - to trans -Golgi network results in the synthesis of the globo series of glycosphingolipids, whereas vesicular transport is the main source of GlcCer for ganglioside synthesis in the Golgi cisternae. Dual carriageway in the Golgi transport complex Newly synthesized lipids are transported across the Golgi network through vesicular and non-vesicular mechanisms. This study demonstrates that glucosylceramide (GlcCer), which is the common precursor of various glycosphingolipids, is channelled to two topologically distinct pathways in the Golgi. Non-vesicular transport of GlcCer from its site of synthesis in the cis -Golgi to the trans -Golgi results in the synthesis of the globo-series (Gb3) of glycosphingolipids, whereas vesicular transport is the major source of GlcCer for the synthesis of gangliosides in the Golgi cisternae. Newly synthesized proteins and lipids are transported across the Golgi complex via different mechanisms whose respective roles are not completely clear. We previously identified a non-vesicular intra-Golgi transport pathway for glucosylceramide (GlcCer)—the common precursor of the different series of glycosphingolipids—that is operated by the cytosolic GlcCer-transfer protein FAPP2 (also known as PLEKHA8) (ref. 1 ). However, the molecular determinants of the FAPP2-mediated transfer of GlcCer from the cis -Golgi to the trans -Golgi network, as well as the physiological relevance of maintaining two parallel transport pathways of GlcCer—vesicular and non-vesicular—through the Golgi, remain poorly defined. Here, using mouse and cell models, we clarify the molecular mechanisms underlying the intra-Golgi vectorial transfer of GlcCer by FAPP2 and show that GlcCer is channelled by vesicular and non-vesicular transport to two topologically distinct glycosylation tracks in the Golgi cisternae and the trans -Golgi network, respectively. Our results indicate that the transport modality across the Golgi complex is a key determinant for the glycosylation pattern of a cargo and establish a new paradigm for the branching of the glycosphingolipid synthetic pathway.

The molecular machinery responsible for the generation of transport carriers moving from the Golgi complex to the plasma membrane relies on a tight interplay between proteins and lipids. Among the lipid-binding proteins of this machinery, we previously identified the four-phosphate adaptor protein FAPP2, the pleckstrin homology domain of which binds phosphatidylinositol 4-phosphate and the small GTPase ARF1. FAPP2 also possesses a glycolipid-transfer-protein homology domain. Here we show that human FAPP2 is a glucosylceramide-transfer protein that has a pivotal role in the synthesis of complex glycosphingolipids, key structural and signalling components of the plasma membrane. The requirement for FAPP2 makes the whole glycosphingolipid synthetic pathway sensitive to regulation by phosphatidylinositol 4-phosphate and ARF1. Thus, by coupling the synthesis of glycosphingolipids with their export to the cell surface, FAPP2 emerges as crucial in determining the lipid identity and composition of the plasma membrane. FAPP2 is a key component of the glycosphingolipid synthetic pathway, mediating non-vesicular transport of glucosylceramide from its site of synthesis at the cis -Golgi to the trans -Golgi for conversion into complex glycosphingolipids. FAPP2 therefore has an important role in determining the lipid composition of the plasma membrane, which is highly enriched in glycosphingolipids.

Normal growth and development, as well as adaptive responses to various intracellular and environmental stresses, are tightly controlled by transcriptional networks. The evolutionarily conserved genomic sequences across species highlights the architecture of such certain regulatory elements. Among them, one of the most conserved transcription factors is the basic-region leucine zipper (bZIP) family. Herein, we have performed phylogenetic analysis of these bZIP proteins and found, to our surprise, that there exist a few homologous proteins of the family members Jun, Fos, ATF2, BATF, C/EBP and CNC (cap’n’collar) in either viruses or bacteria, albeit expansion and diversification of this bZIP superfamily have occurred in vertebrates from metazoan. Interestingly, a specific group of bZIP proteins is identified, designated Nach (Nrf and CNC homology), because of their strong conservation with all the known CNC and NF-E2 p45 subunit-related factors Nrf1 and Nrf2. Further experimental evidence has also been provided, revealing that Nach1 and Nach2 from the marine bacteria exert distinctive functions, when compared with human Nrf1 and Nrf2, in the transcriptional regulation of antioxidant response element (ARE)-battery genes. Collectively, further insights into these Nach/CNC-bZIP subfamily transcription factors provide a novel better understanding of distinct biological functions of these factors expressed in distinct species from the marine bacteria to humans.

Members of the glycolipid transfer protein superfamily (GLTP) are found from animals and fungi to plants and red micro-alga. Eukaryotes that encode the glucosylceramide synthase responsible for the synthesis of glucosylceramide, the precursor for most glycosphingolipids, also produce GLTPs. Cells that does not synthesize glucosylceramide neither express GLTPs. Based on this genetic relationship there must be a strong correlation between the synthesis of glucosylceramide and GLTPs. To regulate the levels of glycolipids we have used inhibitors of intracellular trafficking, glycosphingolipid synthesis and degradation, and small interfering RNA to down-regulate the activity of glucosylceramide synthase activity. We found that GLTP expression, both at the mRNA and protein levels, is elevated in cells that accumulate glucosylceramide. Monensin and brefeldin A block intracellular vesicular transport mechanisms. Brefeldin A treatment leads to accumulation of newly synthesized glucosylceramide, galactosylceramide and lactosylceramide in a fused endoplasmic reticulum-Golgi complex. On the other hand, inhibiting glycosphingolipid degradation with conduritol-B-epoxide, that generates glucosylceramide accumulation in the lysosomes, did not affect the levels of GLTP. However, glycosphingolipid synthesis inhibitors like PDMP, NB-DNJ and myriocin, all decreased glucosylceramide and GLTP below normal levels. We also found that an 80% loss of glucosylceramide due to glucosylceramide synthase knockdown resulted in a significant reduction in the expression of GLTP. We show here that interfering with membrane trafficking events and simple neutral glycosphingolipid synthesis will affect the expression of GLTP. We postulate that a change in the glucosylceramide balance causes a response in the GLTP expression, and put forward that GLTP might play a role in lipid directing and sensing of glucosylceramide at the ER-Golgi interface.

The glycolipid transfer protein, GLTP, can be found in the cytoplasm, and it has a FFAT-like motif (two phenylalanines in an acidic tract) that targets it to the endoplasmic reticulum (ER). We have previously shown that GLTP can bind to a transmembrane ER protein, vesicle-associated membrane protein-associated protein A (VAP-A), which is involved in a wide range of ER functions. We have addressed the mechanisms that might regulate the association between GLTP and the VAP proteins by studying the capacity of GLTP to recognize different N-linked acyl chain species of glucosylceramide. We used surface plasmon resonance and a lipid transfer competition assay to show that GLTP prefers shorter N-linked fully saturated acyl chain glucosylceramides, such as C8, C12, and C16, whereas long C18, C20, and C24-glucosylceramides are all bound more weakly and transported more slowly than their shorter counterparts. Changes in the intrinsic GLTP tryptophan fluorescence blueshifts, also indicate a break-point between C16- and C18-glucosylceramide in the GLTP sensing ability. It has long been postulated that GLTP would be a sensor in the sphingolipid synthesis machinery, but how this mechanistically occurs has not been addressed before. It is unclear what proteins the GLTP VAP association would influence. Here we found that if GLTP has a bound GlcCer the association with VAP-A is weaker. We have also used a formula for identifying putative FFAT-domains, and we identified several potential VAP-interactors within the ceramide and sphingolipid synthesis pathways that could be candidates for regulation by GLTP.

Ceramides can be delivered to cultured cells without solvents in the form of complexes with cholesteryl phosphocholine. We have analysed the delivery of three different radiolabeled D-erythro-ceramides (C6-Cer, C10-Cer and C16-Cer) to HeLa cells, and followed their metabolism as well as the cell viability. We found that all three ceramides were successfully taken up by HeLa cells when complexed to CholPC in an equimolar ratio, and show that the ceramides show different rates of cellular uptake and metabolic fate. The C6-Cer had the highest incorporation rate, followed by C10-Cer and C16-Cer, respectively. The subsequent effect on cell viability strongly correlated with the rate of incorporation, where C6-Cer had the strongest apoptotic effects. Low-dose (1 μM) treatment with C6-Cer favoured conversion of the precursor to sphingomyelin, whereas higher concentrations (25-100 μM) yielded increased conversion to C6-glucosylceramide. Similar results were obtained for C10-Cer. In the lower-dose C16-Cer experiments, most of the precursor was degraded, whereas at high-dose concentrations the precursor remained un-metabolized. Using this method, we demonstrate that ceramides with different chain lengths clearly exhibit varying rates of cellular uptake. The cellular fate of the externally delivered ceramides are clearly connected to their rate of incorporation and their subsequent effects on cell viability may be in part determined by their chain length.

The glycolipid transfer protein (GLTP) catalyzes the binding and transport of glycolipids, but not phospholipids or neutral lipids. With its all-alpha helical fold, it is the founding member for a new superfamily, however its biological role still remains unclear. We have analyzed changes in the HeLa cell lipidome in response to down- and up-regulation of GLTP expression. We used metabolic labeling and thin layer chromatography analysis, complemented with a lipidomics mass spectroscopic approach. HeLa cells were treated with GLTP siRNA or were transiently overexpressing the GLTP gene. We identified eight different lipid classes that changed as a result of the GLTP down- or up-regulation treatments; glucosylceramide, lactosylceramide, globotriaosylceramide, ceramide, sphingomyelin, cholesterol-esters, diacylglycerol and phosphatidylserine. We discovered that the amount of globotriaosylceramide (Gb3) was extensively lowered after down-regulation of GLTP. Further, an up-regulation of GLTP caused a substantial increase in both the Gb3 and glucosylceramide levels compared to the controls. Total galactosylceramide levels remained unchanged. Both lactosylceramide and ceramide showed small changes, an increase with increasing GLTP and a decrease in the HeLa cell GLTP knockdowns. The cholesterol-esters and diacylglycerol masses increased in cells that had upregulated GLTP protein levels, wheras down-regulation did not affect their amounts. For the glycerophospholipids, phosphatidylserine was the only species that was lower in GLTP overexpressing cells. Phosphatidylethanolamine, phosphatidylglyerol and phosphatidylinositol remained unaltered. A total of 142 lipid species were profiled and quantified using shotgun lipidomics analyses. This work provides for the first time insights into how alternations in the levels of a protein that binds and transfers glycolipids affects the cellular lipid metabolism. We discuss the observed changes in the lipidome and how these relate to GLTP. We suggest, that GLTP not only could be a significant player in cellular sphingolipid metabolism, but also could have a much broader role in the overall lipid metabolism.

[This corrects the article DOI: 10.1371/journal.pone.0070283.].

The UDP - glucose ceramide glucosyltransferase (UGCG) is a key enzyme in the synthesis of glycosylated sphingolipids, since this enzyme generates the precursor for all complex glycosphingolipids (GSL), the GlcCer. The UGCG has been associated with several cancer-related processes such as maintaining cancer stem cell properties or multidrug resistance induction. The precise mechanisms underlying these processes are unknown. Here, we investigated the molecular mechanisms occurring after UGCG overexpression in breast cancer cells. We observed alterations of several cellular properties such as morphological changes, which enhanced proliferation and doxorubicin resistance in UGCG overexpressing MCF-7 cells. These cellular effects seem to be mediated by an altered composition of glycosphingolipid-enriched microdomains (GEMs), especially an accumulation of globotriaosylceramide (Gb3) and glucosylceramide (GlcCer), which leads to an activation of Akt and ERK1/2. The induction of the Akt and ERK1/2 signaling pathway results in an increased gene expression of multidrug resistance protein 1 ( MDR1 ) and anti-apoptotic genes and a decrease of pro-apoptotic gene expression. Inhibition of the protein kinase C (PKC) and phosphoinositide 3 kinase (PI3K) reduced MDR1 gene expression. This study discloses how changes in UGCG expression impact several cellular signaling pathways in breast cancer cells resulting in enhanced proliferation and multidrug resistance.