Search

Lysoplasmalogens are a class of vinyl ether bioactive lipids that have a central role in plasmalogen metabolism and membrane fluidity. The liver X receptor (LXR) transcription factors are important determinants of cellular lipid homeostasis owing to their ability to regulate cholesterol and fatty acid metabolism. However, their role in governing the composition of lipid species such as lysoplasmalogens in cellular membranes is less well studied. Here, we mapped the lipidome of bone marrow-derived macrophages (BMDMs) following LXR activation. We found a marked reduction in the levels of lysoplasmalogen species in the absence of changes in the levels of plasmalogens themselves. Transcriptional profiling of LXR-activated macrophages identified the gene encoding transmembrane protein 86a (TMEM86a), an integral endoplasmic reticulum protein, as a previously uncharacterized sterol-regulated gene. We demonstrate that TMEM86a is a direct transcriptional target of LXR in macrophages and microglia and that it is highly expressed in TREM2 + /lipid-associated macrophages in human atherosclerotic plaques, where its expression positively correlates with other LXR-regulated genes. We further show that both murine and human TMEM86a display active lysoplasmalogenase activity that can be abrogated by inactivating mutations in the predicted catalytic site. Consequently, we demonstrate that overexpression of Tmem86a in BMDM markedly reduces lysoplasmalogen abundance and membrane fluidity, while reciprocally, silencing of Tmem86a increases basal lysoplasmalogen levels and abrogates the LXR-dependent reduction of this lipid species. Collectively, our findings implicate TMEM86a as a sterol-regulated lysoplasmalogenase in macrophages that contributes to sterol-dependent membrane remodeling., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier Inc.)

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., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

Enolase is a glycolytic enzyme known to inhibit cholesteryl ester hydrolases (CEHs). Cholesteryl ester loading of macrophages, as occurs during atherosclerosis, is accompanied by increased Enolase protein and activity. Here, we describe that J774 macrophages treated with LXR agonists exhibit reduced Enolase transcript and protein abundance. Moreover, we show that this reduction is further potentiated by activation of the LXR/RXR heterodimer with the RXR ligand 9-cis retinoic acid. Enolase levels are also reduced in vivo following activation of LXRs in the intestine, but not in the liver. This effect is lost in Lxrαβ-/- mice. In aggregate, our study identified Enolase as a new target of LXRs in vivo, which may promote cholesterol mobilization for subsequent efflux., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Low density lipoprotein (LDL) cholesterol is taken up into cells via clathrin-mediated endocytosis of the LDL receptor (LDLR). Following dissociation of the LDLR-LDL complex, LDL is directed to lysosomes whereas the LDLR recycles to the plasma membrane. Activation of the sterol-sensing nuclear receptors liver X receptors (LXRs) enhances degradation of the LDLR. This depends on the LXR target gene inducible degrader of the LDLR (IDOL), an E3-ubiquitin ligase that promotes ubiquitylation and lysosomal degradation of the LDLR. How ubiquitylation of the LDLR by IDOL controls its endocytic trafficking is currently unknown. Using genetic- and pharmacological-based approaches coupled to functional assessment of LDL uptake, we show that the LXR-IDOL axis targets a LDLR pool present in lipid rafts. IDOL-dependent internalization of the LDLR is independent of clathrin, caveolin, macroautophagy, and dynamin. Rather, it depends on the endocytic protein epsin. Consistent with LDLR ubiquitylation acting as a sorting signal, degradation of the receptor can be blocked by perturbing the endosomal sorting complex required for transport (ESCRT) or by USP8, a deubiquitylase implicated in sorting ubiquitylated cargo to multivesicular bodies. In summary, we provide evidence for the existence of an LXR-IDOL-mediated internalization pathway for the LDLR that is distinct from that used for lipoprotein uptake.

Background/aim: Multidrug Resistance Protein 3 (MRP3) transports bile salts and glucuronide conjugates in vitro and is postulated to protect the liver in cholestasis. Whether the absence of Mrp3 affects these processes in vivo is tested., Methods: Mrp3-deficient mice were generated and the contribution of Mrp3 to bile salt and glucuronide conjugate transport was tested in (1): an Ussing-chamber set-up with ileal explants (2), the liver during bile-duct ligation (3), liver perfusion experiments, and (4) in vitro vesicular uptake experiments., Results: The Mrp3((-/-)) mice show no overt phenotype. No differences between WT and Mrp3-deficient mice were found in the trans-ileal transport of taurocholate. After bile-duct ligation, there were no differences in histological liver damage and serum bile salt levels between Mrp3((-/-)) and WT mice, but Mrp3-deficient mice had lower serum bilirubin glucuronide concentrations. Glucuronide conjugates of hyocholate and hyodeoxycholate are substrates of MRP3 in vitro and in livers that lack Mrp3, there is reduced sinusoidal secretion of hyodeoxycholate-glucuronide after perfusion with hyodeoxycholate., Conclusions: Mrp3 does not have a major role in bile salt physiology, but is involved in the transport of glucuronidated compounds, which could include glucuronidated bile salts in humans.

Several ATP-binding cassette (ABC) transporters can transport drugs out of cells against steep concentration gradients resulting in resistance to the drugs transported. Recent work has shown that at least three members of the family of human Multidrug Resistance-associated Proteins (MRPs), MRP4, 5 and 8, are able to transport some nucleoside-monophosphate analogs. This can result in resistance to the base, nucleoside or nucleotide precursors of these results, at least in cell lines with high levels of transporter. The affinity of these transporters for the nucleotide analogs studied thus far is relatively low (millimolar rather than micromolar), and this limits their potential impact on the resistance. We briefly review how ABC transporters in general, and MRPs in particular, could affect the disposition and cellular accumulation of antiviral compounds.