Cholesterol Pool Research Articles

Mitochondrial respiratory chain dysfunction alters ER sterol sensing and mevalonate pathway activity

Mitochondrial dysfunction induces a strong adaptive retrograde signaling response; however, many of the downstream effectors of this response remain to be discovered. Here, we studied the shared transcriptional responses to three different mitochondrial respiratory chain inhibitors in human primary skin fibroblasts using QuantSeq 3′-RNA-sequencing. We found that genes involved in the mevalonate pathway were concurrently downregulated, irrespective of the respiratory chain complex affected. Targeted metabolomics demonstrated that impaired mitochondrial respiration at any of the three affected complexes also had functional consequences on the mevalonate pathway, reducing levels of cholesterol precursor metabolites. A deeper study of complex I inhibition showed a reduced activity of endoplasmic reticulum–bound sterol-sensing enzymes through impaired processing of the transcription factor Sterol Regulatory Element-Binding Protein 2 and accelerated degradation of the endoplasmic reticulum cholesterol-sensors squalene epoxidase and HMG-CoA reductase. These adaptations of mevalonate pathway activity affected neither total intracellular cholesterol levels nor the cellular free (nonesterified) cholesterol pool. Finally, measurement of intracellular cholesterol using the fluorescent cholesterol binding dye filipin revealed that complex I inhibition elevated cholesterol on intracellular compartments. Taken together, our study shows that mitochondrial respiratory chain dysfunction elevates intracellular free cholesterol levels and therefore attenuates the expression of mevalonate pathway enzymes, which lowers endogenous cholesterol biosynthesis, disrupting the metabolic output of the mevalonate pathway. We conclude that intracellular disturbances in cholesterol homeostasis may alter systemic cholesterol management in diseases associated with declining mitochondrial function.

Annexin A6 and NPC1 regulate LDL-inducible cell migration and distribution of focal adhesions

Cholesterol is considered indispensable for cell motility, but how physiological cholesterol pools enable cells to move forward remains to be clarified. The majority of cells obtain cholesterol from the uptake of Low-Density lipoproteins (LDL) and here we demonstrate that LDL stimulates A431 squamous epithelial carcinoma and Chinese hamster ovary (CHO) cell migration and invasion. LDL also potentiated epidermal growth factor (EGF) -stimulated A431 cell migration as well as A431 invasion in 3-dimensional environments, using organotypic assays. Blocking cholesterol export from late endosomes (LE), using Niemann Pick Type C1 (NPC1) mutant cells, pharmacological NPC1 inhibition or overexpression of the annexin A6 (AnxA6) scaffold protein, compromised LDL-inducible migration and invasion. Nevertheless, NPC1 mutant cells established focal adhesions (FA) that contain activated focal adhesion kinase (pY397FAK, pY861FAK), vinculin and paxillin. Compared to controls, NPC1 mutants display increased FA numbers throughout the cell body, but lack LDL-inducible FA formation at cell edges. Strikingly, AnxA6 depletion in NPC1 mutant cells, which restores late endosomal cholesterol export in these cells, increases their cell motility and association of the cholesterol biosensor D4H with active FAK at cell edges, indicating that AnxA6-regulated transport routes contribute to cholesterol delivery to FA structures, thereby improving NPC1 mutant cell migratory behaviour.

Blocking cholesterol storage to treat Alzheimer's disease.

Cholesterol serves as an essential lipid molecule in various membrane organelles of mammalian cells. The metabolites of cholesterol also play important functions. Acyl-coenzyme A: cholesterol acyltransferase 1 (ACAT1), also named as sterol O-acyltransferase 1, is a membrane-bound enzyme residing at the endoplasmic reticulum (ER). It converts cholesterol to cholesteryl esters (CEs) for storage, and is expressed in all cells. CEs cannot partition in membranes; they can only coalesce as cytosolic lipid droplets. Excess CEs are found in the vulnerable region of the brains of patients with late-onset Alzheimer’s disease (AD), and in cell and mouse models for AD. Reducing CE contents by genetic inactivation of ACAT1, or by pharmacological inhibition of ACAT is shown to reduce amyloidopathy and other hallmarks for AD. To account for the various beneficial actions of the ACAT1 blockade (A1B), a working hypothesis is proposed here: the increase in CE contents observed in the AD brain is caused by damages of cholesterol-rich lipid rafts that are known to occur in neurons affected by AD. These damages cause cholesterol to release from lipid rafts and move to the ER where it will be converted to CEs by ACAT1. In addition, the increase in CE contents may also be caused by overloading with cholesterol-rich substances, or through activation of ACAT1 gene expression by various proinflammatory agents. Both scenarios may occur in microglia of the chronically inflamed brain. A1B ameliorates AD by diverting the cholesterol pool destined for CE biosynthesis such that it can be utilized more efficiently to repair membrane damage in various organelles, and to exert regulatory actions more effectively to defend against AD. To test the validity of the A1B hypothesis in cell culture and in vivo, the current status of various anti-ACAT1 agents that could be further developed is briefly discussed.

Measuring and Manipulating Membrane Cholesterol for the Study of Hedgehog Signaling.

Cholesterol is an abundant lipid in mammalian plasma membranes that regulates the reception of the Hedgehog (Hh) signal in target cells. In vertebrates, cell-surface organelles called primary cilia function as compartments for the propagation of Hh signals. Recent structural, biochemical, and cell-biological studies have led to the model that Patched-1 (PTCH1), the receptor for Hh ligands, uses its transporter-like activity to lower cholesterol accessibility in the membrane surrounding primary cilia. Cholesterol restriction at cilia may represent the long-sought-after mechanism by which PTCH1 inhibits Smoothened (SMO), a cholesterol-responsive transmembrane protein of the G protein-coupled receptor superfamily that transmits the Hh signal across the membrane.Protein probes based on microbial cholesterol-binding proteins revealed that PTCH1 controls only a subset of the total cholesterol molecules, a biochemically defined fraction called accessible cholesterol. The accessible cholesterol pool coexists (and exchanges) with a pool of sequestered cholesterol, which is bound to phospholipids like sphingomyelin. In this chapter, we describe how to measure the accessible and sequestered cholesterol pools in live cells with protein-based probes. We discuss how to purify and fluorescently label these probes for use in flow cytometry and microscopy-based measurements of the cholesterol pools. Additionally, we describe how to modulate accessible cholesterol levels to determine if this pool regulates Hh signaling (or any other cellular process of interest).

Induction of microRNA hsa-let-7d-5p, and repression of HMGA2, contribute protection against lipid accumulation in macrophage ‘foam’ cells

Accumulation of excess cholesterol and cholesteryl ester in macrophage ‘foam’ cells within the arterial intima characterises early ‘fatty streak’ atherosclerotic lesions, and is accompanied by epigenetic changes, including altered expression of microRNA sequences which determine of gene and protein expression. This study established that exposure to lipoproteins, including acetylated LDL, induced macrophage expression of microRNA hsa-let-7d-5p, a sequence previously linked with tumour suppression, and repressed expression of one of its target genes, high mobility group AT hook 2 (HMGA2). A let-7d-5p mimic repressed expression of HMGA2 (18%; p < 0.05) while a marked increase (2.9-fold; p < 0.05) in expression of HMGA2 was noted in the presence of let-7d-5p inhibitor. Under these conditions, let-7d-5p mimic significantly (p < 0.05) decreased total (10%), free (8%) and cholesteryl ester (21%) mass, while the inhibitor significantly (p < 0.05) increased total (29%) and free cholesterol (29%) mass, compared with the relevant controls. Let-7d-5p inhibition significantly (p < 0.05) increased endogenous biosynthesis of cholesterol (38%) and cholesteryl ester (39%) pools in macrophage ‘foam’ cells, without altering the cholesterol efflux pathway, or esterification of exogenous radiolabelled oleate. Let-7d-5p inhibition in sterol-loaded cells increased the level of HMGA2 protein (32%; p < 0.05), while SiRNA knockdown of this protein (29%; p < 0.05) resulted in a (21%, p < 0.05) reduction in free cholesterol mass. Thus, induction of let-7d-5p, and repression of its target HMGA2, in macrophages is a protective response to the challenge of increased cholesterol influx into these cells; dysregulation of this response may contribute to atherosclerosis and other disorders such as cancer.

Cholesterol as a Second Messenger in the Hedgehog Signaling Pathway

The Hedgehog (HH) pathway is a cell-cell communication system essential for embryonic development; loss of proper HH signaling results in birth defects and cancer. Signaling is initiated when HH ligands are received on target cells by their receptor, Patched-1 (PTCH1). This relieves the inhibitory effect of PTCH1 on the G protein-coupled receptor Smoothened (SMO), allowing the HH signal to be transmitted across the membrane. The question of how PTCH1 inhibits SMO has remained a central mystery in developmental signaling for 25 years. Prior work implicated cholesterol as an endogenous lipid regulator of SMO. However, a major conundrum is presented by the abundance of cholesterol: how can a lipid that makes up 30%-50% of the plasma membrane be used to regulate a key signaling pathway? Using a custom CRISPR library focused on genes involved in lipid regulation, we asked the more general question of which lipids regulate HH signaling in target cells. We found that Sphingomyelin (SM) levels negatively regulate HH signaling. We show that SM exerts its effect by sequestering cholesterol into complexes, altering the chemical activity (or “accessibility”) of cholesterol. This highlights a key concept in membrane biology: only the accessible pool of cholesterol is available to interact with proteins and engage in signaling reactions. Using toxin-based sensors to measure accessible and sequestered cholesterol in cells, we find that HH ligands change cholesterol accessibility selectively in the membrane of the primary cilium. Primary cilia are antenna-like organelles required for HH signaling in all vertebrates. By compartmentalizing HH signaling in cilia, cells can use accessible cholesterol to communicate between PTCH1 and SMO without interfering with overall cellular cholesterol homeostasis. Our work suggests that the second messenger that communicates the signal between PTCH1 and SMO is the accessibility of cholesterol in a membrane compartment.

Molecular basis of accessible plasma membrane cholesterol recognition by the GRAM domain of GRAMD1b

Cholesterol is essential for cell physiology. Transport of the “accessible” pool of cholesterol from the plasma membrane (PM) to the endoplasmic reticulum (ER) by ER‐localized GRAMD1 proteins (GRAMD1a/1b/1c) contributes to cholesterol homeostasis. However, how cells detect accessible cholesterol within the PM remains unclear. We show that the GRAM domain of GRAMD1b, a coincidence detector for anionic lipids, including phosphatidylserine (PS), and cholesterol, possesses distinct but synergistic sites for sensing accessible cholesterol and anionic lipids. We find that a mutation within the GRAM domain of GRAMD1b that is associated with intellectual disability in humans specifically impairs cholesterol sensing. In addition, we identified another point mutation within this domain that enhances cholesterol sensitivity without altering its PS sensitivity. Cell‐free reconstitution and cell‐based assays revealed that the ability of the GRAM domain to sense accessible cholesterol regulates membrane tethering and determines the rate of cholesterol transport by GRAMD1b. Thus, cells detect the codistribution of accessible cholesterol and anionic lipids in the PM and fine‐tune the non‐vesicular transport of PM cholesterol to the ER via GRAMD1s.

CHOLESTEROL AND ITS IMPLICATIONS- A REVIEW

Ambiguity associated with cholesterol portrays its significance and concern. Cholesterol is a steroid biomolecule of animal cells with several important functions in living system, such as steroid hormone production, structural architecture of cell membranes, production of vitamin D, sources of bile salts and bile acids. Its sources include endogenous (de novo production by body’s tissues) and exogenous (dietary), contributing to cholesterol pool in the body. Cholesterol homeostasis is essentially regulated by the body. This review focuses on the following; basis of cholesterol, biological functions of cholesterol, structural description of cholesterol, Biosynthesis of cholesterol, cholesterol and its derivatives e.g bile acids, bile salts, steroid hormone, absorption and utilization of dietary cholesterol and current advancement in cholesterol management against risk factors. Hypercholesterolaemia is known to be an important precondition to the etiology of cardiovascular disorders which include atherosclerosis, stroke and coronary heart diseases. Interventions for the management and prevention of hypercholesterolaemia currently advocated include pharmaceuticals (drugs) and non pharmaceuticals, but more concern on non pharmacological therapeutic interventions such as the use of Medicinal Plants and herbs, Nutraceuticals, Diet and Exercises (lifestyle) and Functional and Mediterranean Foods. It is thus glaring that the disease linked implication of cholesterol can be prevented and managed using the appropriate interventions.&#x0D; &#x0D; Peer Review History: &#x0D; Received 4 November 2020; Revised 12 Decembe; Accepted 2 January, Available online 15 January 2021&#x0D; UJPR follows the most transparent and toughest ‘Advanced OPEN peer review’ system. The identity of the authors and, reviewers will be known to each other. This transparent process will help to eradicate any possible malicious/purposeful interference by any person (publishing staff, reviewer, editor, author, etc) during peer review. As a result of this unique system, all reviewers will get their due recognition and respect, once their names are published in the papers. We expect that, by publishing peer review reports with published papers, will be helpful to many authors for drafting their article according to the specifications. Auhors will remove any error of their article and they will improve their article(s) according to the previous reports displayed with published article(s). The main purpose of it is ‘to improve the quality of a candidate manuscript’. Our reviewers check the ‘strength and weakness of a manuscript honestly’. There will increase in the perfection, and transparency.&#x0D; Received file: Comments of reviewer(s): &#x0D; Average Peer review marks at initial stage: 6.0/10&#x0D; Average Peer review marks at publication stage: 8.0/10&#x0D; Reviewer(s) detail:&#x0D; Dr. Rawaa Souhil Al-Kayali, Aleppo University, Syria, rawah67@hotmail.com&#x0D; Dr. U. S. Mahadeva Rao, Universiti Sultan Zainal Abidin, Terengganu Malaysia, raousm@gmail.com&#x0D; Similar Articles:&#x0D; USE OF DIOSGENIN, YAMOGENIN, TIGOGENIN AND NEOTIGOGENIN FOR TREATMENT OF HYPERLIPIDEMIA BY INHIBITING CHOLESTEROL ABSORPTION IN GIT &#x0D;

Imaging cholesterol depletion at the plasma membrane by methyl-β-cyclodextrin

The most common method to modify cellular cholesterol (Chol) content in vitro is to incubate cells with cyclodextrin (CD). However, it is not well understood how Chol is removed by CD. Recent development of protein probes that bind Chol allowed to visualize and to follow cellular Chol semi-quantitatively. In this image, non-toxic Chol-binding D4 fragment of bacterial toxin, perfringolysin O was conjugated with mCherry (mCherry-D4) (1Abe M. Makino A. Hullin-Matsuda F. Kamijo K. Ohno-Iwashita Y. Hanada K. Mizuno H. Miyawaki A. Kobayashi T. A role for sphingomyelin-rich lipid domains in the accumulation of phosphatidylinositol 4,5-bisphosphate to the cleavage furrow during cytokinesis.Mol. Cell Biol. 2012; 32: 1396-1407Crossref PubMed Scopus (98) Google Scholar) and the protein was transiently expressed in HeLa cells for 48 h. Cells were synchronized to mitosis and incubated with and without 1 mM methyl-b-CD (MbCD) in the presence of EGFP-D4 in the medium. Selected images are shown. Accumulated images are shown in Supplemental videos 1 and 2. Average fluorescence intensities of EGFP-D4 (green) and mCherry-D4 (magenta) were quantified in the area surrounded by the white rectangle and schematically shown in the kymographs. The quantitated data are shown as graphs in Supplemental Fig. 1. Supplemental Fig. 2 shows that equivalent amount of EGFP-D4 and mCherry-D4 displayed similar Chol concentration dependent binding to phosphatidylcholine/Chol membranes. Under this condition, mCherry-D4 and EGFP-D4 bind accessible Chol (2Das A. Brown M.S. Anderson D.D. Goldstein J.L. Radhakrishnan A. Three pools of plasma membrane cholesterol and their relation to cholesterol homeostasis.Elife. 2014; 3e02882Crossref Scopus (171) Google Scholar) in the inner and outer leaflet of the plasma membrane, respectively (1Abe M. Makino A. Hullin-Matsuda F. Kamijo K. Ohno-Iwashita Y. Hanada K. Mizuno H. Miyawaki A. Kobayashi T. A role for sphingomyelin-rich lipid domains in the accumulation of phosphatidylinositol 4,5-bisphosphate to the cleavage furrow during cytokinesis.Mol. Cell Biol. 2012; 32: 1396-1407Crossref PubMed Scopus (98) Google Scholar). Due to high threshold of Chol-detection by D4, intracellular organelles were not labeled with mCherry-D4 and depletion of Chol was accompanied by the intracellular accumulation of the protein. MbCD induced the rapid decrease (5-15 min) of inner leaflet Chol below the D4-binding threshold followed by the slow depletion phase in both outer and inner leaflets. The interaction between D4 or MbCD and cultured cells is not fully elucidated. Although MbCD is endocytosed, endocytosis is inhibited in mitotic cells (3Kobayashi T. Pagano R.E. Lipid transport during mitosis. Alternative pathways for delivery of newly synthesized lipids to the cell surface.J. Biol. Chem. 1989; 264: 5966-5973Abstract Full Text PDF PubMed Google Scholar). Binding of acrylodan-labeled D4 to PC/Chol membrane is inhibited by cytosol (4Courtney K.C. Fung K.Y. Maxfield F.R. Fairn G.D. Zha X. Comment on 'Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol'.Elife. 2018; 7e38493Crossref PubMed Scopus (16) Google Scholar). In addition to Chol, MbCD is reported to interact with other lipids and protein components of the membrane. However, extraction of cytosolic protein by MbCD has not been reported. Our result suggests that the removal of outer leaflet Chol by MbCD is accompanied by the rapid flip of Chol from the inner leaflet to the outer leaflet. However, we cannot exclude the possibility that MbCD preferentially removed Chol from the inner leaflet. EQUIPMENT: FV 1000 confocal microscope with a 60x 1.1 NA Plan Apo objective lens (Olympus) equipped with an environmental chamber maintained with humidity, 37°C, and 5% CO2. ImageQuant LAS 500 (Cytiva). REAGENTS: DMEM supplemented with 10% fetal bovine serum, Lipofectamine 3000 (Thermo Fisher Scientific), pET28/His6-EGFP-D4 (RIKEN BRC, Gene Engineering Division, Catalog # RDB13961), pET28/His6-mCherry-D4 (RIKEN BRC, Catalog # RDB14300), pDEST/mCherry-D4 (1), nocodazole (Sigma-Aldrich), MβCD (Cyclolab), SYPRO Ruby (Thermo Fisher Scientific). This article contains supplemental data. The authors declare that they have no conflicts of interest with the contents of this article. We are grateful to Dr. Françoise Hullin-Matsuda from INSA Lyon for critically reading the manuscript. M. A. conceptualization, methodology, investigation, data curation, writing-original draft preparation, reviewing, and editing. T. K. conceptualization, writing-original draft preparation, reviewing, and editing.

The use of anthrolysin O and ostreolysin A to study cholesterol in cell membranes.

Cholesterol is a major component of the plasma membranes (PMs) of animal cells, comprising 35-40mol% of total PM lipids. Recent studies using cholesterol-binding bacterial toxins such as domain 4 of Anthrolysin O (ALOD4) and fungal toxins such as Ostreolysin A (OlyA) have revealed new insights into the organization of PM cholesterol. These studies have defined three distinct pools of PM cholesterol-a fixed pool that is essential for membrane integrity, a sphingomyelin (SM)-sequestered pool that can be detected by OlyA, and a third pool that is accessible and can be detected by ALOD4. Accessible cholesterol is available to interact with proteins and transport to the endoplasmic reticulum (ER), and controls many cellular signaling processes including cholesterol homeostasis, Hedgehog signaling, and bacterial and viral infection. Here, we provide detailed descriptions for the use of ALOD4 and OlyA, both of which are soluble and non-lytic proteins, to study cholesterol organization in the PMs of animal cells. Furthermore, we describe two new versions of ALOD4 that we have developed to increase the versatility of this probe in cellular studies. One is a dual His6 and FLAG epitope-tagged version and the other is a fluorescent version where ALOD4 is fused to Neon, a monomeric fluorescent protein. These new forms of ALOD4 together with previously described OlyA provide an expanded collection of tools to sense, visualize, and modulate levels of accessible and SM-sequestered cholesterol on PMs and study the role of these cholesterol pools in diverse membrane signaling events.

Evaluation of the available cholesterol concentration in the inner leaflet of the plasma membrane of mammalian cells

Cholesterol is an essential component of the mammalian plasma membrane involved in diverse cellular processes. Our recent quantitative imaging analysis using ratiometric cholesterol sensors showed that the available cholesterol concentration in the inner leaflet of the plasma membrane (IPM) is low in unstimulated cells and increased in a stimulus-specific manner to trigger cell signaling events. However, the transbilayer distribution of cholesterol in the plasma membrane of mammalian cells remains controversial. Here we report a systematic and rigorous evaluation of basal IPM cholesterol levels in a wide range of mammalian cells with different properties employing cholesterol sensors derived from the D4 domain of the Perfringolysin O toxin and a sterol-transfer protein, Osh4. Results consistently showed that, although basal IPM cholesterol levels vary significantly among cells, they remain significantly lower than cholesterol levels in the outer leaflets. We found that IPM cholesterol levels were particularly low in all tested primary cells. These results support the universality of the low basal IPM cholesterol concentration under physiological conditions. We also report here the presence of sequestered IPM cholesterol pools, which may become available to cytosolic proteins under certain physiological conditions. We hypothesize that these pools may partly account for the low basal level of available IPM cholesterol. In conclusion, we provide new experimental data that confirm the asymmetric transbilayer distribution of the plasma membrane cholesterol, which may contribute to regulation of various cellular signaling processes at the plasma membrane.

A Novel Pool of Microparticle Cholesterol Is Elevated in Rheumatoid Arthritis but Not in Systemic Lupus Erythematosus Patients

Microparticles are sub-micron, membrane-bound particles released from virtually all cells and which are present in the circulation. In several autoimmune disorders their amount and composition in the circulation is altered. Microparticle surface protein expression has been explored as a differentiating tool in autoimmune disorders where the clinical pictures can overlap. Here, we examine the utility of a novel lipid-based marker—microparticle cholesterol, present in all microparticles regardless of cellular origin—to distinguish between rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). We first isolated a series of microparticle containing lipoprotein deficient fractions from patient and control plasma. There were no significant differences in the size, structure or protein content of microparticles isolated from each group. Compared to controls, both patient groups contained significantly greater amounts of platelet and endothelial cell-derived microparticles. The cholesterol content of microparticle fractions isolated from RA patients was significantly greater than those from either SLE patients or healthy controls. Our data indicate that circulating non-lipoprotein microparticle cholesterol, which may account for 1–2% of measured cholesterol in patient samples, may represent a novel differentiator of disease, which is independent of cellular origin.

Cholesterol access in cellular membranes controls Hedgehog signaling.

The Hedgehog (Hh) signaling pathway coordinates cell-cell communication in development and regeneration. Defects in this pathway underlie diseases ranging from birth defects to cancer. Hh signals are transmitted across the plasma membrane by two proteins, Patched 1 (PTCH1) and Smoothened (SMO). PTCH1, a transporter-like tumor-suppressor protein, binds to Hh ligands, but SMO, a G-protein-coupled-receptor family oncoprotein, transmits the Hh signal across the membrane. Recent structural, biochemical and cell-biological studies have converged at the surprising model that a specific pool of plasma membrane cholesterol, termed accessible cholesterol, functions as a second messenger that conveys the signal between PTCH1 and SMO. Beyond solving a central puzzle in Hh signaling, these studies are revealing new principles in membrane biology: how proteins respond to and remodel cholesterol accessibility in membranes and how the cholesterol composition of organelle membranes is used to regulate protein function.

Lactobacillus plantarum 06CC2 reduces hepatic cholesterol levels and modulates bile acid deconjugation in Balb/c mice fed a high-cholesterol diet.

Previous study suggested that dietary intake of Lactobacillus plantarum 06CC2 (LP06CC2) isolated from Mongolian dairy products showed various health beneficial effects. Here, the effect of LP06CC2 on the cholesterol metabolism in mice fed a cholesterol‐loaded diet was evaluated. Cholesterol and LP06CC2 were incorporated into the AIN93G‐based diet to evaluate the effect on cholesterol metabolism in Balb/c mice. Serum and liver cholesterol levels were significantly increased in mice fed a cholesterol‐loaded diet whereas the LP06CC2 ingestion suppressed the increase of liver cholesterol. LP06CC2 suppressed the increase of the hepatic damage indices. The increase of the cecal content and fecal butyrate were observed in mice fed LP06CC2. The analysis of bile acids clearly showed that LP06CC2 increased their deconjugation indicating the decrease of bile acid absorption. The protein expression of hepatic Cyp7A1 was also suppressed by LP06CC2 in mice fed cholesterol. Finally, in vitro studies showed that LP06CC2 had the most potent ability to deconjugate bile acids using glycocholate among the tested probiotic lactic acid bacteria isolated from Mongolian dairy products. Taken together, LP06CC2 is a promising microorganism for the reduction of the cholesterol pool via modulation of bile acid deconjugation.

Aster Proteins Regulate the Accessible Cholesterol Pool in the Plasma Membrane.

Recent studies have demonstrated the existence of a discrete pool of cholesterol in the plasma membranes (PM) of mammalian cells-referred to as the accessible cholesterol pool-that can be detected by the binding of modified versions of bacterial cytolysins (e.g., anthrolysin O). When the amount of accessible cholesterol in the PM exceeds a threshold level, the excess cholesterol moves to the endoplasmic reticulum (ER), where it regulates the SREBP2 pathway and undergoes esterification. We reported previously that the Aster/Gramd1 family of sterol transporters mediates nonvesicular movement of cholesterol from the PM to the ER in multiple mammalian cell types. Here, we investigated the PM pool of accessible cholesterol in cholesterol-loaded fibroblasts with a knockdown of Aster-A and in mouse macrophages from Aster-B and Aster-A/B-deficient mice. Nanoscale secondary ion mass spectrometry (NanoSIMS) analyses revealed expansion of the accessible cholesterol pool in cells lacking Aster expression. The increased accessible cholesterol pool in the PM was accompanied by reduced cholesterol movement to the ER, evidenced by increased expression of SREBP2-regulated genes. Cosedimentation experiments with liposomes revealed that the Aster-B GRAM domain binds to membranes in a cholesterol concentration-dependent manner and that the binding is facilitated by the presence of phosphatidylserine. These studies revealed that the Aster-mediated nonvesicular cholesterol transport pathway controls levels of accessible cholesterol in the PM, as well as the activity of the SREBP pathway.

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This article refers to:Aster Proteins Regulate the Accessible Cholesterol Pool in the Plasma Membrane

The Alzheimer's disease‐associated C99 fragment of APP regulates cellular cholesterol trafficking

The link between cholesterol homeostasis and cleavage of the amyloid precursor protein (APP), and how this relationship relates to Alzheimer's disease (AD) pathogenesis, is still unknown. Cellular cholesterol levels are regulated through crosstalk between the plasma membrane (PM), where most cellular cholesterol resides, and the endoplasmic reticulum (ER), where the protein machinery that regulates cholesterol levels resides. The intracellular transport of cholesterol from the PM to the ER is believed to be activated by a lipid‐sensing peptide(s) in the ER that can cluster PM‐derived cholesterol into transient detergent‐resistant membrane domains (DRMs) within the ER, also called the ER regulatory pool of cholesterol. When formed, these cholesterol‐rich domains in the ER maintain cellular homeostasis by inducing cholesterol esterification as a mechanism of detoxification while attenuating its de novo synthesis. In this manuscript, we propose that the 99‐aa C‐terminal fragment of APP (C99), when delivered to the ER for cleavage by γ‐secretase, acts as a lipid‐sensing peptide that forms regulatory DRMs in the ER, called mitochondria‐associated ER membranes (MAM). Our data in cellular AD models indicates that increased levels of uncleaved C99 in the ER, an early phenotype of the disease, upregulates the formation of these transient DRMs by inducing the internalization of extracellular cholesterol and its trafficking from the PM to the ER. These results suggest a novel role for C99 as a mediator of cholesterol disturbances in AD, potentially explaining early hallmarks of the disease.

MITOCHONDRIAL CHOLESTEROL AND CANCER.

Cholesterol is a crucial component of membrane bilayers that determines their physical and functional properties. Cells largely satisfy their need for cholesterol through the novo synthesis from acetyl-CoA and this demand is particularly critical for cancer cells to sustain dysregulated cell proliferation. However, the association between serum or tissue cholesterol levels and cancer development is not well established as epidemiologic data do not consistently support this link. While most preclinical studies focused on the role of total celular cholesterol, the specific contribution of the mitochondrial cholesterol pool to alterations in cancer cell biology has been less explored. Although low compared to other bilayers, the mitochondrial cholesterol content plays an important physiological function in the synthesis of steroid hormones in steroidogenic tissues or bile acids in the liver and controls mitochondrial function. In addition, mitochondrial cholesterol metabolism generates oxysterols, which in turn, regulate multiple pathways, including cholesterol and lipid metabolism as well as cell proliferation. In the present review, we summarize the regulation of mitochondrial cholesterol, including its role in mitochondrial routine performance, cell death and chemotherapy resistance, highlighting its potential contribution to cancer. Of particular relevance is hepatocellular carcinoma, whose incidence in Western countries had tripled in the past decades due to the obesity and type II diabetes epidemic. A better understanding of the role of mitochondrial cholesterol in cancer development may open up novel opportunities for cancer therapy.

Cholesterol access aids pathogen spread.

Some cytosolic bacteria deform the plasma membrane to spread from cell to cell. Secretion of 25-hydroxy-cholesterol by macrophages has emerged as a protective response that depletes the cholesterol pool used by these bacteria to disseminate through epithelia.

Interferon-mediated reprogramming of membrane cholesterol to evade bacterial toxins.

Plasma membranes of animal cells are enriched for cholesterol. Cholesterol-dependent cytolysins (CDCs) are pore forming toxins secreted by bacteria that target membrane cholesterol for their effector function. Phagocytes are essential for clearance of CDC-producing bacteria; however, mechanisms these cells use to evade the deleterious effects of CDCs are largely unknown. Here, we report that interferon (IFN) signals convey resistance to CDC-induced pores on macrophages and neutrophils. We traced IFN-mediated resistance to CDCs to the rapid modulation of a specific pool of cholesterol in the plasma membrane of macrophages, without changing total cholesterol levels. Resistance to CDC pore formation requires production of the oxysterol 25-hydroxycholesterol, inhibition of cholesterol synthesis, and redistribution of cholesterol to an esterified cholesterol pool. Accordingly, blocking IFN’s ability to reprogram cholesterol metabolism abrogates cellular protection and renders mice more susceptible to CDC-induced tissue damage. These studies illuminate targeted regulation of membrane cholesterol content as a host-defense strategy.