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Simple Summary: Epithelial ovarian cancer (EOC) is the most lethal gynaecological cancer. Over 80% of cases have already spread at diagnosis, and these patients face a five-year survival rate of 35%. EOC cells often spread to the greater omentum, an abdominal fat pad. Here, EOC cells take-up cholesterols. Excessive amounts of cholesterol are lethal; thus, we proposed that the ABCA1 cholesterol transporter exports cholesterol from serous EOC cells to maintain cholesterol balance. Indeed, we found that reducing the level of ABCA1 could suppress serous EOC growth in two-dimensional as well as three-dimensional cell culture and also hindered their migration, a key process required for cancer spread. We also identified drugs that impair EOC cell growth by inhibiting cholesterol export. Our data demonstrate that disrupting the cholesterol balance by targeting ABCA1 may be an effective treatment strategy for EOC patients. Background: Epithelial ovarian cancer (EOC) is the most lethal gynaecological malignancy with over 80% of cases already disseminated at diagnosis and facing a dismal five-year survival rate of 35%. EOC cells often spread to the greater omentum where they take-up cholesterol. Excessive amounts of cholesterol can be cytocidal, suggesting that cholesterol efflux through transporters may be important to maintain homeostasis, and this may explain the observation that high expression of the ATP-binding cassette A1 (ABCA1) cholesterol transporter has been associated with poor outcome in EOC patients. Methods: ABCA1 expression was silenced in EOC cells to investigate the effect of inhibiting cholesterol efflux on EOC biology through growth and migration assays, three-dimensional spheroid culture and cholesterol quantification. Results: ABCA1 suppression significantly reduced the growth, motility and colony formation of EOC cell lines as well as the size of EOC spheroids, whilst stimulating expression of ABCA1 reversed these effects. In serous EOC cells, ABCA1 suppression induced accumulation of cholesterol. Lowering cholesterol levels using methyl-B-cyclodextrin rescued the effect of ABCA1 suppression, restoring EOC growth. Furthermore, we identified FDA-approved agents that induced cholesterol accumulation and elicited cytocidal effects in EOC cells. Conclusions: Our data demonstrate the importance of ABCA1 in maintaining cholesterol balance and malignant properties in EOC cells, highlighting its potential as a therapeutic target for this disease. [ABSTRACT FROM AUTHOR]

Research demonstrates that 7-ketocholesterol inhibits cholesterol efflux in all cellular systems studied. Data suggest that the structure of 7-ketocholesterol and its effect on the membrane lipid composition may be the key regulatory elements influencing its action.

The relationship existing between the lipid content and size of discoidal reconstituted HDL particles and their ability to promote cellular cholesterol efflux is analyzed. The particles are shown to act as efficient acceptors of the cell cholesterol in the presence of cell models, ABCA1 and ABCG1.

A study is conducted to investigate the phosphorylation of apolipoprotein (apoE) by CK2 in vitro and to map potential phsophorylation sites. The phosphorylation of apoE by CK2 as well as the activation of CK2 by apoE is likely to be relevant in vivo where apoE, CK2, and tau are co-localized with additional CK2 targets on neuronal microtubules.

A heritage building and its site present a range of challenges when developing preventive conservation strategies for the collection in the building. These challenges originate from the need to preserve the authenticity and physical integrity of the building and site while simultaneously addressing the environmental vulnerabilities of the objects in the collection. To address these challenges, preventive conservation strategies must be specific to each heritage building and the collection in the building, rather than based on a set of prescriptive rules. With heritage buildings, preventive conservation of the collections must balance environmental vulnerabilities of the collections with the available causative factors presented by the context, the ability of the building envelope and its systems to mediate these causative factors, and the capacity of the institution for sustained implementation of the preventive strategy. The authors present an approach to the resolution of these competing or conflicting challenges. This approach engages the preventive conservator, the building specialist, and the responsible institutional steward in a collaborative effort, informed by a systemic understanding of the issues, in developing a realistic and achievable preventive conservation strategy. [ABSTRACT FROM AUTHOR]

Sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) is a recently identified phosphodiesterase, which is a secreted N-linked glycoprotein. SMPDL3A is highly homologous to acid sphingomyelinase (aSMase), but unlike aSMase cannot cleave sphingomyelin. Rather, SMPDL3A hydrolyzes nucleotide tri- and diphosphates and their derivatives. While recent structural studies have shed light on these unexpected substrate preferences, many other aspects of SMPDL3A biology, which may give insight into its function in vivo, remain obscure. Here, we investigate the roles of N-glycosylation in the expression, secretion and activity of human SMPDL3A, using inhibitors of N-glycosylation and site-directed mutagenesis, with either THP-1 macrophages or CHO cells expressing human SMPDL3A. Tunicamycin (TM) treatment resulted in expression of non-glycosylated SMPDL3A that was not secreted, and was largely degraded by the proteasome. Proteasomal inhibition restored levels of SMPDL3A in TM-treated cells, although this non-glycosylated protein lacked phosphodiesterase activity. Enzymatic deglycosylation of purified recombinant SMPDL3A also resulted in significant loss of phosphodiesterase activity. Site-directed mutagenesis of individual N-glycosylation sites in SMPDL3A identified glycosylation of Asn69 and Asn222 as affecting maturation of its N-glycans and secretion. Glycosylation of Asn356 in SMPDL3A, an N-linked site conserved throughout the aSMase-like family, was critical for protection against proteasomal degradation and preservation of enzymatic activity. We provide the first experimental evidence for a predicted 22 residue N-terminal signal peptide in SMPDL3A, which is essential for facilitating glycosylation and is removed from the mature protein secreted from CHO cells. In conclusion, site-specificN-glycosylation is essential for the intracellular stability, secretion and activity of human SMPDL3A. [ABSTRACT FROM AUTHOR]

Epidemiologic studies have shown an inverse correlation between high-density lipoprotein (HDL) cholesterol (HDL-C) levels and cardiovascular disease outcomes. However, the hypothesis of a causal relationship between HDL-C and cardiovascular disease has been challenged by genetic and clinical studies. Serum cholesterol efflux capacity (CEC) is an important measure of HDL function in humans. Recent large clinical studies have shown a correlation between in vitro CEC and cardiovascular disease prevalence and incidence, which appears to be independent of HDL-C concentration. The present review summarizes recent large clinical studies and introduces important methodological considerations. Further studies are required to standardize and establish the reproducibility of this measure of HDL function and clarify whether modulating CEC will emerge as a useful therapeutic target. [ABSTRACT FROM AUTHOR]

Dynamins are fission proteins that mediate endocytic and exocytic membrane events and are pharmacological therapeutic targets. These studies investigate whether dynamin II regulates constitutive protein secretion and show for the first time that pharmacological inhibition of dynamin decreases secretion of apolipoprotein E (apoE) and several other proteins constitutively secreted from primary human macrophages. Inhibitors that target recruitment of dynamin to membranes (MiTMABs) or directly target the GTPase domain (Dyngo or Dynole series), dose- and time- dependently reduced the secretion of apoE. SiRNA oligo’s targeting all isoforms of dynamin II confirmed the involvement of dynamin II in apoE secretion. Inhibition of secretion was not mediated via effects on mRNA or protein synthesis. 2D-gel electrophoresis showed that inhibition occurred after apoE was processed and glycosylated in the Golgi and live cell imaging showed that inhibited secretion was associated with reduced post-Golgi movement of apoE-GFP-containing vesicles. The effect was not restricted to macrophages, and was not mediated by the effects of the inhibitors on microtubules. Inhibition of dynamin also altered the constitutive secretion of other proteins, decreasing the secretion of fibronectin, matrix metalloproteinase 9, Chitinase-3-like protein 1 and lysozyme but unexpectedly increasing the secretion of the inflammatory mediator cyclophilin A. We conclude that pharmacological inhibitors of dynamin II modulate the constitutive secretion of macrophage apoE as a class effect, and that their capacity to modulate protein secretion may affect a range of biological processes. [ABSTRACT FROM AUTHOR]

Cholesterol excess is typical of various diseases including atherosclerosis. We have investigated whether cholesterol accumulation in the ER (endoplasmic reticulum) can inhibit exit of vesicular cargo and secretion of proteins by studying apoE (apolipoprotein E), a significant glycoprotein in human health and disease. CHO (Chinese hamster ovary) cells expressing human apoE under a cholesterol-independent promoter incubated with cholesterol-cyclodextrin complexes showed increased levels of cellular free and esterified cholesterol, inhibition of SREBP-2 (sterol-regulatory-element-binding protein 2) processing, and a mild induction of ERstress, indicating significant accumulation of cholesterol in the ER. Secretion of apoE was markedly inhibited by cholesterol accumulation, and similar effects were observed in cells enriched with lipoprotein-derived cholesterol and in primary human macrophages. Removal of excess cholesterol by a cyclodextrin vehicle restored apoE secretion, indicating that the transport defect was reversible. That cholesterol impaired protein trafficking was supported by the cellular accumulation of less sialylated apoE glycoforms, and by direct visualization of altered ER to Golgi transport of thermo-reversible VSVG (vesicular stomatitis virus glycoprotein) linked to GFP (green fluorescent protein). We conclude that intracellular accumulation of cholesterol in the ER reversibly inhibits protein transport and secretion. Strategies to correct ER cholesterol may restore homoeostatic processes and intracellular protein transport in conditions characterized by cholesterol excess. [ABSTRACT FROM AUTHOR]

The biochemical composition and biophysical properties of cell membranes are hypothesized to affect cellular processes such as phagocytosis. Here, we examined the plasma membranes of murine macrophage cell lines during the early stages of uptake of immunoglobulin G (IgG)-coated polystyrene particles. We found that the plasma membrane undergoes rapid actin-independent condensation to form highly ordered phagosomal membranes, the biophysical hallmark of lipid rafts. Surprisingly, these membranes are depleted of cholesterol and enriched in sphingomyelin and ceramide. Inhibition of sphingomyelinase activity impairs membrane condensation, F-actin accumulation at phagocytic cups and particle uptake. Switching phagosomal membranes to a cholesterol-rich environment had no effect on membrane condensation and the rate of phagocytosis. In contrast, preventing membrane condensation with the oxysterol 7-ketocholesterol, even in the presence of ceramide, blocked F-actin dissociation from nascent phagosomes and particle uptake. In conclusion, our results suggest that ordered membranes function to co-ordinate F-actin remodelling and that the biophysical properties of phagosomal membranes are essential for phagocytosis. [ABSTRACT FROM AUTHOR]

Caveolin-1 (Cav1), a structural protein required for the formation of invaginated membrane domains known as caveolae, has been implicated in cholesterol trafficking and homeostasis. Here we investigated the contribution of Cav1 to apolipoprotein A-I (apoA-I) cell surface binding and intracellular processing using mouse embryonic fibroblasts (MEFs) derived from wild type (WT) or Cav1-deficient (Cav1-/-) animals. We found that cells expressing Cav1 have 2.6-fold more apoA-I binding sites than Cav1-/- cells although these additional binding sites are not associated with detergent-free lipid rafts. Further, Cav1- mediated binding targets apoA-I for internalization and degradation and these processes are not correlated to cholesterol efflux. Despite lower apoA-I binding, cholesterol efflux from Cav1-/- MEFs is 1.7-fold higher than from WT MEFs. Stimulation of ABCA1 expression with an LXR agonist enhances cholesterol efflux from both WT and Cav1-/- cells without increasing apoA-I surface binding or affecting apoA-I processing. Our results indicate that there are at least two independent lipid binding sites for apoA-I; Cav1-mediated apoA-I surface binding and uptake is not linked to cholesterol efflux, indicating that membrane domains other than caveolae regulate ABCA1-mediated cholesterol efflux. [ABSTRACT FROM AUTHOR]

Abstract: Oxysterols are structurally identical to cholesterol, but with one or more additional oxygen containing functional groups (such as alcohol, carbonyl or epoxide groups). The wide array of oxysterols encountered in human health and disease vary in their origin (either enzymic or non-enzymic), and their putative effects and/or function(s). Some are thought to be damaging, whereas others may play important physiological roles, including in the regulation of cholesterol homeostasis. In this review, we will concentrate on the major cellular oxysterols. We summarise their location, generation, metabolism and elimination, as well as providing insights into the latest research into their regulatory roles in cholesterol homeostasis. [Copyright &y& Elsevier]

The T lymphocyte plasma membrane condenses at the site of activation but the functional significance of this receptormediated membrane reorganization is not yet known. Here we demonstrate that membrane condensation at the T cell activation sites can be inhibited by incorporation of the oxysterol 7-ketocholesterol (7KC), which is known to prevent the formation of raft-like liquid-ordered domains in model membranes. We enriched T cells with 7KC, or cholesterol as control, to assess the importance of membrane condensation for T cell activation. Upon 7KC treatment, T cell antigen receptor (TCR) triggered calcium fluxes and early tyrosine phosphorylation events appear unaltered. However, signaling complexes form less efficiently on the cell surface, fewer phosphorylated signaling proteins are retained in the plasma membrane and actin restructuring at activation sites is impaired in 7KC-enriched cells resulting in compromised downstream activation responses. Our data emphasizes lipids as an important medium for the organization at T cell activation sites and strongly indicates that membrane condensation is an important element of the T cell activation process. [ABSTRACT FROM AUTHOR]

The article discusses the coexistence of foam cells and hypocholesterolemia in mice lacking the ABC transporters A1 and G1. It explains the relation of targeted disruption of both ABCA1 and ABCG1 in mice, despite severe plasma hypocholesterolemia, to massive lipid accumulation and foam cell formation of tissue macrophages. It was stated that ABCA1 and ABCG1 was important in the prevention of foam cell formation.