Haploid genetic screens identify SPRING/C12ORF49 as a determinant of SREBP signaling and cholesterol metabolism

The sterol-regulatory element binding proteins (SREBP) are central transcriptional regulators of lipid metabolism. Using haploid genetic screens we identify the SREBPRegulating Gene (SPRING/C12ORF49) as a determinant of the SREBP pathway. SPRING is a glycosylated Golgi-resident membrane protein and its ablation in Hap1 cells, Hepa1-6 hepatoma cells, and primary murine hepatocytes reduces SREBP signaling. In mice, Spring deletion is embryonic lethal yet silencing of hepatic Spring expression also attenuates the SREBP response. Mechanistically, attenuated SREBP signaling in SPRING(KO) cells results from reduced SREBP cleavage-activating protein (SCAP) and its mislocalization to the Golgi irrespective of the cellular sterol status. Consistent with limited functional SCAP in SPRING(KO) cells, reintroducing SCAP restores SREBP-dependent signaling and function. Moreover, in line with the role of SREBP in tumor growth, a wide range of tumor cell lines display dependency on SPRING expression. In conclusion, we identify SPRING as a previously unrecognized modulator of SREBP signaling

A Specific Activity-Based Probe to Monitor Family GH59 Galactosylceramidase, the Enzyme Deficient in Krabbe Disease

Galactosylceramidase (GALC) is the lysosomal β-galactosidase responsible for the hydrolysis of galactosylceramide. Inherited deficiency in GALC causes Krabbe disease, a devastating neurological disorder characterized by accumulation of galactosylceramide and its deacylated counterpart, the toxic sphingoid base galactosylsphingosine (psychosine). We report the design and application of a fluorescently tagged activity-based probe (ABP) for the sensitive and specific labeling of active GALC molecules from various species. The probe consists of a β-galactopyranose-configured cyclophellitol-epoxide core, conferring specificity for GALC, equipped with a BODIPY fluorophore at C6 that allows visualization of active enzyme in cells and tissues. Detection of residual GALC in patient fibroblasts holds great promise for laboratory diagnosis of Krabbe disease. We further describe a procedure for in situ imaging of active GALC in murine brain by intra-cerebroventricular infusion of the ABP. In conclusion, this GALC-specific ABP should find broad applications in diagnosis, drug development, and evaluation of therapy for Krabbe disease

Glucosylated cholesterol in mammalian cells and tissues: formation and degradation by multiple cellular β-glucosidases.

The membrane lipid glucosylceramide (GlcCer) is continuously formed and degraded. Cells express two GlcCer-degrading β-glucosidases, glucocerebrosidase (GBA) and GBA2, located in and outside the lysosome, respectively. Here we demonstrate that through transglucosylation both GBA and GBA2 are able to catalyze in vitro the transfer of glucosyl-moieties from GlcCer to cholesterol, and vice versa. Furthermore, the natural occurrence of 1-O-cholesteryl-β-D-glucopyranoside (GlcChol) in mouse tissues and human plasma is demonstrated using LC-MS/MS and (13)C6-labeled GlcChol as internal standard. In cells, the inhibition of GBA increases GlcChol, whereas inhibition of GBA2 decreases glucosylated sterol. Similarly, in GBA2-deficient mice, GlcChol is reduced. Depletion of GlcCer by inhibition of GlcCer synthase decreases GlcChol in cells and likewise in plasma of inhibitor-treated Gaucher disease patients. In tissues of mice with Niemann-Pick type C disease, a condition characterized by intralysosomal accumulation of cholesterol, marked elevations in GlcChol occur as well. When lysosomal accumulation of cholesterol is induced in cultured cells, GlcChol is formed via lysosomal GBA. This illustrates that reversible transglucosylation reactions are highly dependent on local availability of suitable acceptors. In conclusion, mammalian tissues contain GlcChol formed by transglucosylation through β-glucosidases using GlcCer as donor. Our findings reveal a novel metabolic function for GlcCer.This study was made possible by the ERC AdG CHEMBIOSPHIN. The authors declare no financial conflicts of interest relevant to this study

The glucosylceramide synthase inhibitor N-(5-adamantane-1-yl-methoxy-pentyl)-deoxynojirimycin induces sterol regulatory element-binding protein-regulated gene expression and cholesterol synthesis in HepG2 cells

Recent findings have implicated glycosphingolipids as modulators of insulin receptor activity. Studies with C57BL/6J ob/ob mice have shown that insulin sensitivity is enhanced by the synthetic hydrophobic iminosugar N-(5-adamantane-1-yl-methoxy-pentyl)- deoxynojirimycin (AMP-DNM) that inhibits glucosylceramide synthase. Here, we treated the liver hepatoma cell line HepG2 with AMP-DNM, resulting in a 70% reduction of glycosphingolipids, and we analyzed the effect on gene expression. Using whole human genome 44K oligonucleotide arrays, we identified 89 genes that were significantly (p <0.01) up-or down-regulated by AMP-DNM treatment. Of the 56 up-regulated genes, 17 were direct target genes for transcription factors sterol regulatory element-binding protein (SREBP) 1 or SREBP2, which activate genes in the sterol biosynthesis pathway. An increase in cholesterol production rate confirmed that the induction of SREBP target genes seen at the mRNA level resulted in activation of the cholesterol biosynthesis pathway. It is interesting to note that the cholesterol content of the cells did not increase. It is noteworthy that no effects were found on expression of genes related to cell receptor signaling pathways, neither on toxicity nor cell growth. Our findings indicate that inhibition of glucosylceramide synthase with AMP-DNM leads to activation of SREBP target genes and synthesis of cholesterol in HepG2 cell

Differential use of E2 ubiquitin conjugating enzymes for regulated degradation of the rate-limiting enzymes HMGCR and SQLE in cholesterol biosynthesis

Background and aims: Cholesterol is an essential lipid for cellular function and membrane integrity, and hence its cellular levels and distribution must be tightly regulated. Biosynthesis of cholesterol is ramped when its cellular levels are low. Herein, the ER-resident and rate-limiting enzymes 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and squalene monooxygenase (SQLE) play a prominent role. We have recently reported that MARCH6, an E3 ubiquitin ligase, specifically promotes cholesterol-stimulated ubiquitylation and subsequent proteasomal degradation of SQLE, but not of HMGCR. To further delineate how post-translational regulation of SQLE and HMGCR is differentially achieved, we hypothesized that their sterol-dependent degradation machinery makes use of distinct E2 ubiquitin conjugating enzymes. Methods: To study this possibility, we therefore used a CRISPR/Cas9-based approach to screen for ER-associated degradation (ERAD)-associated E2 enzymes that are essential for MARCH6-dependent degradation of SQLE. Results: We report here the identification of UBE2J2 as the primary E2 ubiquitin conjugating enzyme essential for this process in mammalian cells, in contrast to UBE2G2, which is essential for sterol-stimulated degradation of HMGCR. We demonstrate that ablating UBE2J2 disturbs cholesterol-accelerated SQLE degradation in multiple human cell types, including cells of hepatic origin, and that the ability of UBE2J2 to support SQLE degradation critically depends on its enzymatic activity. Conclusions: Our findings establish UBE2J2 as an important partner of MARCH6 in cholesterol-stimulated degradation of SQLE, thereby contributing to the complex regulation of cellular cholesterol homeostasis

Very low serum adiponectin levels in patients with type 1 Gaucher disease without overt hyperglycemia

Gaucher disease (glucocerebrosidase deficiency) is characterized by massive accumulation of lipid-laden macrophages in various tissues. Patients with Gaucher disease show a hitherto unexplained increase in hepatic glucose output. Because adiponectin is thought to influence hepatic glucose output, we studied its serum concentration in a cohort of patients with Gaucher disease. Serum adiponectin was indeed found to be markedly reduced in patients (median value, 3.1 microg/mL; range, 1.4-6.3 microg/mL) as compared with healthy subjects (median value 5.6 microg/mL range, 1.9-14.0 microg/mL). Successful treatment of patients was accompanied by an increase in serum adiponectin, from 3.1 to 3.6 microg/mL (P = .002). In healthy individuals, low levels of circulating adiponectin are generally associated with obesity. In patients with Gaucher disease, however, adiponectin levels did not correlate with body mass index. The hypoadiponectinemia in Gaucher patients is most likely attributable to their low-grade chronic inflammation. The characteristic storage macrophages produce inflammatory cytokines such as tumor necrosis factor alpha that is known to suppress adiponectin production. It is of interest that the very low adiponectin levels in Gaucher patients are not accompanied by hyperglycemia, contrary to their effect in obese individuals. It is hypothesized that the excessive hepatic glucose production in these patients balances the assumed increased glucose consumption by the massive amounts of storage macrophages. Hypoadiponectemia may play a regulatory role in preventing hypoglycemia in this conditio

Curdlan-mediated regulation of human phagocyte-specific chitotriosidase

Human phagocyte-specific chitotriosidase is part of innate immunity and shows anti-fungal activity towards chitin-containing fungi. We investigated the effect of stimulation of the C-type lectin receptor dectin-1 by beta-1,3-glucan (curdlan) on chitotriosidase expression and release by human phagocytes. We observed that curdlan triggers chitotriosidase release from human neutrophils. In addition, we show that curdlan impairs chitotriosidase induction in monocytes. Finally, curdlan temporarily induces chitotriosidase in enzyme-expressing monocyte-derived macrophages, followed by reduction of chitotriosidase expression after prolonged stimulation. These data on regulation of phagocyte-specific chitotriosidase following curdlan recognition support an important role of chitotriosidase in the elimination of chitin-containing pathogens. (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserve

Regulation of intestinal LDLR by the LXR-IDOL axis

Background and aims: Cholesterol metabolism is tightly regulated by transcriptional and post-transcriptional mechanisms. Accordingly, dysregulation of cholesterol metabolism is a major risk factor for the development of coronary artery disease and associated complications. In recent years, it has become apparent that next to the liver, the intestine plays a key role in systemic cholesterol metabolism by governing cholesterol absorption, secretion, and incorporation into lipoprotein particles. We have previously demonstrated that the Liver X receptor (LXR)-regulated E3 ubiquitin ligase inducible degrader of LDLR (IDOL) is a regulator of cholesterol uptake owing to its ability to promote the ubiquitylation of the low-density lipoprotein receptor (LDLR). However, whether the LXR-IDOL-LDLR axis regulates the LDLR in the intestine and whether this influences intestinal cholesterol homeostasis is not known. Methods: In this study, we evaluated the role of the LXR-IDOL-LDLR axis in enterocyte cell models and in primary enterocytes isolated from Idol(−/−) and wild type mice. Furthermore, we studied the regulation of intestinal LDLR in Idol(−/−) and in wild type mice treated with the LXR agonist GW3965. Finally, we assessed ezetimibe-induced trans-intestinal cholesterol efflux in Idol(−/−) mice. Results: We show that in a wide range of intestinal cell lines LXR activation decreases LDLR protein abundance, cell surface occupancy, and LDL uptake in an IDOL-dependent manner. Similarly, we find that pharmacological dosing of C57BL6/N mice with the LXR agonist GW3965 increases Idol expression across the intestine with a concomitant reduction in Ldlr protein. Conversely, primary enterocytes isolated from Idol(−/−) mice have elevated Ldlr. To test whether these changes contribute to trans-intestinal cholesterol efflux, we measured fecal cholesterol in mice following ezetimibe dosing, but found no differences between Idol(−/−) and control mice in this setting. Conclusions: In conclusion, our study establishes that the LXR-IDOL-LDLR axis is active in the intestine and is part of the molecular circuitry that maintains cholesterol homeostasis in enterocytes

Identification of the ER-resident E3 ubiquitin ligase RNF145 as a novel LXR-regulated gene

Cellular cholesterol metabolism is subject to tight regulation to maintain adequate levels of this central lipid molecule. Herein, the sterol-responsive Liver X Receptors (LXRs) play an important role owing to their ability to reduce cellular cholesterol load. In this context, identifying the full set of LXR-regulated genes will contribute to our understanding of their role in cholesterol metabolism. Using global transcriptional analysis we report here the identification of RNF145 as an LXR-regulated target gene. We demonstrate that RNF145 is regulated by LXRs in both human and mouse primary cells and cell lines, and in vivo in mice. Regulation of RNF145 by LXR depends on a functional LXR-element in its proximal promotor. Consistent with LXR-dependent regulation of Rnf145 we show that regulation is lost in macrophages and fibroblasts from Lxrαβ(-/-) mice, and also in vivo in livers of Lxrα(-/-) mice treated with the LXR synthetic ligand T0901317. RNF145 is closely related to RNF139/TRC8, an E3 ligase implicated in control of SREBP processing. However, silencing of RNF145 in HepG2 or HeLa cells does not impair SREBP1/2 processing and sterol-responsive gene expression in these cells. Similar to TRC8, we demonstrate that RNF145 is localized to the ER and that it possesses intrinsic E3 ubiquitin ligase activity. In summary, we report the identification of RNF145 as an ER-resident E3 ubiquitin ligase that is transcriptionally controlled by LX