Cholesterol Binding Research Articles

Effects of sulfated modification on the physicochemical properties and biological activities of β-glucans from Qingke (Tibetan hulless barley)

In this study, the reaction conditions of sulfur trioxide-pyridine (SO3-Pyr) method for the modification of Qingke β-glucans (THB) were optimized by response surface methodology, and effects of different degrees of substitution (low, medium, and high) on the physicochemical properties, antioxidant activities, and in vitro hypolipidemic activities of THB were investigated. The optimal reaction conditions to obtain the high degree of substitution of sulfated β-glucans were as follows: ratio of SO3-Pyr to THB of 16.88 g/g, reaction time of 2.03 h, and reaction temperature of 57.54 °C. Results showed that sulfated modification significantly affected the water solubilities, apparent viscosities, molecular weights, and molar ratios of constituent monosaccharides of THB. Besides, the sulfated THB exhibited much better antioxidant activities (DPPH and nitric oxide radical scavenging activities, and reducing powers), in vitro binding properties (fat, cholesterol, and bile-acid binding capacities), and pancreatic lipase inhibition activities than that of THB. Indeed, the sulfated THB with higher degree of substitution has stronger antioxidant activities and in vitro hypolipidemic activities. Results suggested that the sulfated modification could be an efficient approach for the improvement of functional properties of THB, and sulfated THB could be further explored as functional food ingredients for industrial applications.

Physical changes, laboratory parameters, and bone mineral density during testosterone treatment in adolescents with gender dysphoria

IntroductionCurrent treatment guidelines for adolescents with gender dysphoria recommend therapy with gonadotropin-releasing hormone agonists (GnRHa) and testosterone in transgender males. However, most evidence on the safety and efficacy of testosterone is based on studies in adults. AimThis study aimed to investigate the efficacy and safety of testosterone treatment in transgender adolescents. MethodsThe study included 62 adolescents diagnosed with gender dysphoria who had started GnRHa treatment and had subsequently received testosterone treatment for more than 6 months. Main Outcome MeasureVirilization, anthropometry, laboratory parameters, and bone mineral density (BMD) were analyzed. ResultsAdolescents were treated with testosterone for a median duration of 12 months. Voice deepening began within 3 months in 85% of adolescents. Increased hair growth was first reported on the extremities, followed by an increase of facial hair. Acne was most prevalent between 6 and 12 months of testosterone therapy. Most adolescents had already completed linear growth; body mass index and systolic blood pressure increased but diastolic blood pressure did not change. High-density lipoprotein (HDL) cholesterol and sex hormone binding globulin significantly decreased, but hematocrit, hemoglobin, prolactin, androstenedione, and dehydroepiandrosterone sulfate significantly increased, although not all changes were clinically significant. Other lipids and HbA1c did not change. Vitamin D deficiency was seen in 32–54% throughout treatment. BMD z-scores after 12 to 24 months of testosterone treatment remained below z-scores before the start of GnRHa treatment. Clinical ImplicationsAdolescents need to be counseled about side effects with potential longer term implications such as increased hematocrit and decreased HDL cholesterol and decreased BMD z-scores. They should be advised on diet, including adequate calcium and vitamin D intake; physical exercise; and the use of tobacco and alcohol to avoid additional risk factors for cardiovascular disease and osteoporosis. Strengths & LimitationsStrengths are the standardized treatment regimen and extensive set of safety parameters investigated. Limitations are the limited duration of follow-up and lack of a control group so some of the observed changes may be due to normal maturation rather than to treatment. ConclusionTestosterone effectively induced virilization beginning within 3 months in the majority of adolescents. Acne was a common side effect, but no short-term safety issues were observed. The increased hematocrit, decreased HDL cholesterol, and decreased BMD z-scores are in line with previous studies. Further follow-up studies will need to establish if the observed changes result in adverse outcomes in the long term.Stoffers IE, de Vries MC, Hannema SE. Physical Changes, Laboratory Parameters, and Bone Mineral Density During Testosterone Treatment in Adolescents with Gender Dysphoria. J Sex Med 2019;16:1459–1468.

Cholesterol Binding to the Transmembrane Region of a Group 2 Hemagglutinin (HA) of Influenza Virus Is Essential for Virus Replication, Affecting both Virus Assembly and HA Fusion Activity.

Hemagglutinin (HA) of influenza virus is incorporated into cholesterol-enriched nanodomains of the plasma membrane. Phylogenetic group 2 HAs contain the conserved cholesterol consensus motif (CCM) YKLW in the transmembrane region. We previously reported that mutations in the CCM retarded intracellular transport of HA and decreased its nanodomain association. Here, we analyzed whether cholesterol interacts with the CCM. Incorporation of photocholesterol into HA was significantly reduced if the whole CCM is replaced by alanine, both using immunoprecipitated HA and when HA is embedded in the membrane. We next used reverse genetics to investigate the significance of the CCM for virus replication. No virus was rescued if the whole motif is exchanged (YKLW4A); singly (LA) or doubly (YK2A and LW2A) mutated virus showed decreased titers and a comparative fitness disadvantage. In polarized cells, transport of HA mutants to the apical membrane was not disturbed. Reduced amounts of HA and cholesterol were incorporated into the viral membrane. Mutant viruses exhibit a decrease in hemolysis, which is only partially corrected if the membrane is replenished with cholesterol. More specifically, viruses have a defect in hemifusion, as demonstrated by fluorescence dequenching. Cells expressing HA YKLW4A fuse with erythrocytes, but the number of events is reduced. Even after acidification unfused erythrocytes remain cell bound, a phenomenon not observed with wild-type HA. We conclude that cholesterol binding to a group 2 HA is essential for virus replication. It has pleiotropic effects on virus assembly and membrane fusion, mainly on lipid mixing and possibly a preceding step.IMPORTANCE The glycoprotein HA is a major pathogenicity factor of influenza viruses. Whereas the structure and function of HA's ectodomain is known in great detail, similar data for the membrane-anchoring part of the protein are missing. Here, we demonstrate that the transmembrane region of a group 2 HA interacts with cholesterol, the major lipid of the plasma membrane and the defining element of the viral budding site nanodomains of the plasma membrane. The cholesterol binding motif is essential for virus replication. Its partial removal affects various steps of the viral life cycle, such as assembly of new virus particles and their subsequent cell entry via membrane fusion. A cholesterol binding pocket in group 2 HAs might be a promising target for a small lipophilic drug that inactivates the virus.

CRISPR-Cas9 Mediated TSPO Gene Knockout alters Respiration and Cellular Metabolism in Human Primary Microglia Cells.

The 18 kDa translocator protein (TSPO) is an evolutionary conserved cholesterol binding protein localized in the outer mitochondrial membrane. It has been implicated in the regulation of various cellular processes including oxidative stress, proliferation, apoptosis, and steroid hormone biosynthesis. Since the expression of TSPO in activated microglia is upregulated in various neuroinflammatory and neurodegenerative disorders, we set out to examine the role of TSPO in an immortalized human microglia C20 cell line. To this end, we performed a dual approach and used (i) lentiviral shRNA silencing to reduce TSPO expression, and (ii) the CRISPR/Cas9 technology to generate complete TSPO knockout microglia cell lines. Functional characterization of control and TSPO knockdown as well as knockout cells, revealed only low de novo steroidogenesis in C20 cells, which was not dependent on the level of TSPO expression or influenced by the treatment with TSPO-specific ligands. In contrast to TSPO knockdown C20 cells, which did not show altered mitochondrial function, the TSPO deficient knockout cells displayed a significantly decreased mitochondrial membrane potential and cytosolic Ca2+ levels, as well as reduced respiratory function. Performing the rescue experiment by lentiviral overexpression of TSPO in knockout cells, increased oxygen consumption and restored respiratory function. Our study provides further evidence for a significant role of TSPO in cellular and mitochondrial metabolism and demonstrates that different phenotypes of mitochondrial function are dependent on the level of TSPO expression.

Modulation of Cholesterol-Dependent Activity of Macrophages IC-21 by a Peptide Containing Two CRAC-Motifs from Protein M1 of Influenza Virus

Functioning of many membrane proteins is regulated by cholesterol. Some proteins were shown to contain cholesterol-recognizing amino-acid consensus (CRAC) motif. In particular, influenza virus protein M1 bears these motifs. To study functional roles and action mechanisms of peptides containing cholesterol-recognizing amino-acid consensus (CRAC) motifs, we designed and synthesized peptide RTKLWEMLVELGNMDKAVKLWRKLKR (peptide P4), which includes two peptides corresponding to CRAC-containing α-helices 3 and 6 of influenza virus protein M1 (LEVLMEWLKTR, aa 39–49, and NNMDKAVKLYRKLK, aa 91–105, respectively). We show that peptide P4 in a concentration range from 0.5 to 50 μM dose-dependently modulates cholesterol-dependent interactions of cultured macrophages IC-21 with 2-micron latex particles mimicking bacteria. The effect of peptide P4 was stronger than the effects of the composing peptides studied previously and exhibited a biphasic dose–response relationship. At concentrations from 0.5 to 10 μM peptide P4 significantly increased the number of cell-associated particles; at concentrations 10–20 μM the number of cell-associated particles decreased, and at concentrations 20–50 μM peptide P4 suppressed the cell activity and cell adhesion and produced a toxic effect. Upon extraction of membrane cholesterol by means of methyl-β-cyclodextrin the inhibitory effects of peptide P4 developed at much lower concentrations. The biphasic dose-dependence of the peptide P4 effect and the influence of methyl-β-cyclodextrin can be explained by the ability of peptide P4 to interact with membrane cholesterol. We suggest that peptide P4 at low concentrations facilitates the formation of cholesterol-enriched domains in plasma membrane and thus stimulates aggregation of cell receptors responsible for interactions of macrophages with particles. At higher concentrations peptide P4 may interfere with the activity of cholesterol-dependent receptors due to competitive binding of cholesterol and thus suppress both cell–particle and cell–substrate interactions. We believe that peptide P4 may serve as a useful tool for studies of cholesterol-dependent processes in cells and may serve a basis for the design of new antimicrobial and immunomodulating substances.

Modulation of Transmembrane Domain Interactions in Neu Receptor Tyrosine Kinase by Membrane Fluidity and Cholesterol.

The activation mechanism of the ErbB family of receptors is of considerable medical interest as they are linked to a number of human cancers, including an aggressive form of breast cancer. In the rat analogue of the human ErbB2 receptor, referred to as Neu, a point mutation in the transmembrane domain (V664E) has been shown to trigger oncogenic transformation. While the structural impact of this mutation has been widely studied in the past to yield models for the active state of the Neu receptor, little is known about the impact of cholesterol on its structure. Given previous reports of the influence of cholesterol on other receptor tyrosine kinases (RTKs), as well as the modulation of lipid composition in cancer cells, we wished to investigate how cholesterol content impacts the structure of the Neu transmembrane domain. We utilized high-resolution magic angle spinning solid-state NMR to measure 13C-13C coupling of selectively labelled probe residues in the Neu transmembrane domain in lipid bilayers containing cholesterol. We observe inter-helical coupling between residues that support helix-helix interactions on both dimerization motifs reported in the literature (A661-XXX-G665 and I659-XXX-V663). We further explore how changes in cholesterol concentration alter transmembrane domain interactions and the properties and mechanics of the bilayer. We interpret our results in light of previous studies relating RTK activity to cholesterol enrichment and/or depletion, and propose a novel model to explain our data that includes the recognition and binding of cholesterol by the Neu transmembrane domain through a putative cholesterol-recognition/interaction amino acid consensus sequence.

The cholesterol transfer protein GRAMD1A regulates autophagosome biogenesis.

Autophagy mediates the degradation of damaged proteins, organelles and pathogens, and plays a key role in health and disease. Thus, the identification of new mechanisms involved in the regulation of autophagy is of major interest. In particular, little is known about the role of lipids and lipid-binding proteins in the early steps of autophagosome biogenesis. Using target-agnostic, high-content, image-based identification of indicative phenotypic changes induced by small molecules, we have identified autogramins as a new class of autophagy inhibitor. Autogramins selectively target the recently discovered cholesterol transfer protein GRAM domain-containing protein 1A (GRAMD1A, which had not previously been implicated in autophagy), and directly compete with cholesterol binding to the GRAMD1A StART domain. GRAMD1A accumulates at sites of autophagosome initiation, affects cholesterol distribution in response to starvation and is required for autophagosome biogenesis. These findings identify a new biological function of GRAMD1A and a new role for cholesterol in autophagy.

Multiple neurosteroid and cholesterol binding sites in voltage-dependent anion channel-1 determined by photo-affinity labeling

Voltage-dependent anion channel-1 (VDAC1) is a mitochondrial porin that is implicated in cellular metabolism and apoptosis, and modulated by numerous small molecules including lipids. VDAC1 binds sterols, including cholesterol and neurosteroids such as allopregnanolone. Biochemical and computational studies suggest that VDAC1 binds multiple cholesterol molecules, but photolabeling studies have identified only a single cholesterol and neurosteroid binding site at E73. To identify all the binding sites of neurosteroids in VDAC1, we apply photo-affinity labeling using two sterol-based photolabeling reagents with complementary photochemistry: 5α-6-AziP which contains an aliphatic diazirine, and KK200 which contains a trifluoromethyl-phenyldiazirine (TPD) group. 5α-6-AziP and KK200 photolabel multiple residues within an E73 pocket confirming the presence of this site and mapping sterol orientation within this pocket. In addition, KK200 photolabels four other sites consistent with the finding that VDAC1 co-purifies with five cholesterol molecules. Both allopregnanolone and cholesterol competitively prevent photolabeling at E73 and three other sites indicating that these are common sterol binding sites shared by both neurosteroids and cholesterol. Binding at the functionally important residue E73 suggests a possible role for sterols in regulating VDAC1 signaling and interaction with partner proteins.

Deciphering the Allosteric Effect of Antagonist Vismodegib on Smoothened Receptor Deactivation Using Metadynamics Simulation.

The smoothened receptor (Smo) plays a key role in Hedgehog (Hh) signaling pathway and it has been regarded as an efficacious therapeutic target for basal cell carcinoma (BCC) and medulloblastoma (MB). Nevertheless, the resistance mutation and active mutants of Smo have put forward the requirement of finding more effective inhibitors. Herein, we performed metadynamics simulations on Smo bound with vismodegib (Smo-Vismod) and with cholesterol (Smo-CLR), respectively, to explore the inhibition mechanism of vismodegib. The simulation results indicated that vismodegib-induced shifts of TM5, TM6, and TM7, which permitted the extracellular extension of TM6 and extracellular loop3 (ECL3) to enter the extracellular cysteine-rich domain (CRD) groove. Therefore, an open CRD groove that has not been noticed previously was observed in Smo-Vismod complex. As a consequence, the occupied CRD groove prevents the binding of cholesterol. In addition, the HD and ECLs play crucial roles in the interaction of CRD and TMD. These results reveal that TM5, TM6, and TM7 play important roles in allosteric inhibition the activation of Smo and disrupting cholesterol binding by vismodegib binding. Our results are expected to contribute to understanding the allosteric inhibition mechanism of Smo by vismodegib. Moreover, the detailed conformational changes contribute to the development of novel Smo inhibitors against resistance mutation and active mutants of Smo.

Physicochemical characteristics and biological activities of polysaccharides from the leaves of different loquat (Eriobotrya japonica) cultivars

In order to explore polysaccharides from loquat leaves (LLPs) as functional food ingredients for prevention and treatment of obesity and type 2 diabetes, the physicochemical characteristics, in vitro binding properties, and inhibitory effects on α-amylase and α-glucosidase of polysaccharides from the leaves of different loquat cultivars, including ‘Baiyu’, ‘Chuannong8’, ‘Yuanbao’, and ‘Dawuxing’, were investigated and compared. Results showed that the contents of total uronic acids and degree of esterification in LLPs ranged from (27.04 ± 1.76)% to (41.46 ± 1.91)%, and from (20.54 ± 0.11)% to (26.90 ± 0.10)%, respectively. The weight-average molecular weights of LLPs ranged from 4.307 × 106 Da to 5.101 × 106 Da, and the major constituent monosaccharides of LLPs were rhamnose, galacturonic acid, arabinose, and galactose. Furthermore, LLPs exerted strong in vitro fat binding, cholesterol binding, and bile acid binding capacities, as well as remarkable inhibitory effects on α-amylase and α-glucosidase, which might be attributed to their molecular weights, molecular weight distributions, and degree of esterification. Results are helpful for better understanding of chemical structures and bioactivities of polysaccharides from loquat leaves, and LLPs had good potential to be explored as functional food ingredients for prevention and treatment of obesity and type 2 diabetes.

Influence of prior pre-treatments on molecular structure and digestibility of succinylated foxtail millet starch

Influence of different pre-treatments (Ultra-sonication, heat-moisture treatment (HMT) and acid hydrolysis) on the functional properties, in vitro binding capacity and structural properties of succinylated foxtail millet starch were investigated. The degree of substitution was found to be higher in the succinylated starch pretreated by sonication (USFS-0.39). Pre-treatment of succinylated starch by citric acid (ASFS) had markedly increased the resistant starch content. In vitro studies have confirmed that USFS exhibited the highest cholesterol binding capacity at both pH 2.0 (1.86 mg/g) and pH 7.0 (3.12 mg/g) owing to the chemical bonding and entrapment of cholesterol molecules in the starch matrix. FT-IR had confirmed the cross-linking of succinic anhydride as indicated by the presence of a new peak at 1724 cm−1. This study concludes that prior treatment of succinylated starch by ultra-sonication is an efficient way to improve the resistant starch content and principally in vitro cholesterol and bile acid/salts binding ability.

MHC class II conformers drive differential B cell activation

Abstract MHC class II molecules present exogenous antigen-derived peptides to CD4 T cells to elicit T cell-based help/regulation. While these peptides represent a robust source of heterogeneity, the class II molecules themselves are also heterogeneous. MHC class II molecules exist in two distinct conformational states, based on alternative pairing and differential cholesterol binding of the molecule’s α and β chain’s transmembrane domains. What have been termed “M2 paired” class II (M2 class II) represents the majority of cell surface class II, whereas M1 paired class II (M1 class II) is a smaller fraction of surface molecules. However, M1 class II is enriched in “lipid raft” membrane domains, is highly stimulatory for CD4 T cells and is selectively linked to B cell activation signaling pathways. Moreover, in B lymphocytes M1 class II selectively associates with intracellular antigen-B cell receptor (BCR) complexes and is selectively loaded with peptide derived from the processing of BCR-bound cognate antigen. In contrast, peptides from non-cognate antigen internalized by fluid-phase endocytosis (as would happen for self-antigens) is loaded onto both M1 and M2 class II. Here, we show that while selective engagement of M1 class II drives B cell calcium signaling (an activation event), co-engagement of M1 and M2 class II allows an M2 class II-associated inhibitory activity to block M1 class II signaling. These findings provide new insight into the molecular mechanisms of MHC class II signaling by revealing the distinct signaling properties of M1 and M2 class II. The results also suggest a B cell-intrinsic mechanism by which T cell recognition of M1 vs. M2 peptide-class II complexes could regulate B cell activation via differential class II signaling.

SUN-042 Loss of the 18-Kda Translocator Protein Tspo Destabilizes Cholesterol Homeostasis in a Hepatic Steatosis Cell Model

The 18-kDa translocator protein TSPO is a conserved ubiquitous high affinity cholesterol binding protein localized in the outer mitochondrial membrane. It plays a critical role in the segregation and transport of free cholesterol (FC) from the outer to the inner mitochondrial membrane in steroidogenic cells. Numerous studies have shown the presence of TSPO in liver. Imaging studies in humans, using specific TSPO ligands, showed that TSPO is highly expressed and accurately mirrors the histological picture of non-alcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH). The function of TSPO in simple steatosis and NASH is unknown. Wild type Huh7 (WT) is a well-differentiated human hepatocyte-derived carcinoma cell line, which contains TSPO in mitochondria. In the present study, we generated a stable TSPO knockdown (TSPO KD) in the Huh7 cell line. To mimic hepatic steatosis in vitro, we treated WT and TSPO KD cells with either acetylated LDL (AcLDL), to deliver cholesterol into the cells, or AcLDL plus 58035, an Acetyl-CoA Acetyltransferase (ACAT) and thus cholesterol esterification inhibitor, to induce the accumulation of FC. AcLDL treatment significantly increased FC levels in WT cells; this effect was more pronounced in TSPO KD cells. In contrast, treatment of the cells with AcLDL and 58035 resulted in reduced FC levels likely due to the damage induced by FC accumulation that led to reduced cell proliferation. Considering that TSPO binds FC with high affinity, we investigated the underlying mechanism/link between TSPO KD and FC accumulation by examining the conversion between FC and cholesterol ester (CE) upon AcLDL treatment. The results obtained showed that knocking down TSPO expression reduced ACAT2 (transforming FC to CE) levels, but not cholesterol ester hydrolase (CEH) converting CE to FC, expression. TSPO was found to directly interact with ACAT2. Under these conditions, the accumulation of FC led to the appearance of lipid droplets (LD) of increased size in TSPO KD compared to WT cells, suggesting that TSPO plays a role in LD formation and/or dynamics in liver cells. GRP78, IRE1A, and PERK endoplasmic reticulum stress markers were also elevated in TSPO KD cells following AcLDL treatment while the mitochondrial membrane potential decreased compared to WT cells. Moreover, increased numbers of lysosomes and autophagosomes were observed in TSPO KD cells, an effect further potentiated by the treatment with AcLDL plus 58035. This finding was validated by the up regulation of the LAMP1 and LC3B, lysosome and autophagic structure markers, respectively. Taken together, these data suggest that under cholesterol treatment, TSPO loss would trigger ER stress, mitochondrial dysfunction, and lipid droplet accumulation via FC accumulation, and ultimately cause intracellular protein and organelle degradation, hallmarks of hepatic steatosis.

In vitro assessment of hypocholesterolemic activity of Lactococcus lactis subsp. lactis

BackgroundCholesterol removal by isolated Lactococcus lactis subsp. lactis was investigated in vitro. The studied mechanisms were (1) assimilation of cholesterol by growing bacterial cells, (2) deconjugation of bile salts by bile salt hydrolase, (3) precipitation of cholesterol with deconjugated bile, (4) binding of cholesterol to the cell membrane, and (5) incorporation of cholesterol into the cell membrane.ResultLevels of cholesterol assimilated by growing cells (43.70%) were significantly higher than those assimilated by resting cells (12.93%) or counterpart dead cells (6.35%). Scanning electron micrographs indicated that cholesterol adsorbed to the bacterial cell wall. A comparison of IR spectra of Lactococcus lactis subsp. lactis grown with and without cholesterol using an FTIR spectroscopy database revealed intensity changes associated with sulfamide bonds, P–H stretching and the C≡C bonds. Precipitation of cholesterol upon deconjugation of glycocholate was higher (5.53 μg/ml) than that observed upon taurocholate (3.7 μg/ml).ConclusionsThe present study suggested the ability of probiotic Lactococcus lactis subsp. lactis to reduce its cholesterol level through several different mechanisms. Consequently, this probiotic bacterium showed an interested potential of application in cholesterol reduction to be a novel and save prospect in resolving hypocholesteremia and related diseases.

Coffee parchment as a new dietary fiber ingredient: Functional and physiological characterization.

Coffee parchment was evaluated as a potential dietary fiber ingredient. For this purpose, dietary fiber was extracted by enzymatic and non-enzymatic methods and its physicochemical and in vitro hypoglycemic and hypolipidemic properties were investigated. Results revealed that coffee parchment (flakes and flour) was a good source of insoluble dietary fiber (IDF), mainly composed by xylans (35%), lignin (32%), and cellulose (12%). From results, the IDF extraction seemed not to be required the use of enzymes. Coffee parchment did not stand out by its content of phenolic compounds and antioxidant capacity, but milling process improved them. Due to its physical structure, coffee parchment flakes exhibited high oil holding capacity (3.8 mg L-1), gelation capacity (8%) besides hydration properties, including water holding (3.4 mg L-1), absorption (3.0 mg L-1) and swelling (14 mg L-1) capacities. Its flour and water-insoluble residue showed lower capacities. Nevertheless, these coffee parchment samples presented effective in vitro hypoglycemic properties, showing high glucose adsorption capacity (50-200 mmol L-1), and capacity to decrease its diffusion (13%), and to inhibit α-amylase (52%) that led to lower starch digestibility (until 46%); and also, outstanding in vitro hypolipidemic properties, as inhibition of pancreatic lipase (43%) and binding of cholesterol and sodium cholate (16.6 and 35.3 mg g-1, respectively). These results provide valuable information for the potential use of coffee parchment as new food DF ingredient.

Computational Tools Unravel Putative Sterol Binding Sites in the Lysosomal NPC1 Protein.

Two proteins have been linked as the critical components in the molecular mechanisms involved in the Niemann Pick type C (NPC) disease: NPC1, a 140 kDa polytopic membrane-bound protein, and the smaller (132 residues), water-soluble NPC2 protein. NPC1 is believed to act in tandem with NPC2, transferring cholesterol and other sterols out of the LE/Lys compartments. Mutations in either NPC1 or NPC2 can lead to an accumulation of cholesterol and lipids in the LE/Lys, the primary phenotype of the NPC disease, but approximately 95% of identified disease-causing mutations have been mapped to the membrane-bound NPC1 protein. Here, we investigate the full length, membrane-bound NPC1 protein computationally using a combination of molecular modeling, docking, and molecular dynamics (MD) simulations. An analysis of titratable amino acid side chains, several buried in protein pockets, reveals several nonstandard protonation states for the low-pH scenario (pH 5) that is realized in the lysosome. Together with the location of these buried amino acids, docking studies have identified putative lipid binding domains that are in close proximity to amino acids that, when mutated, are connected to NPC1 loss-of-function. Using energy analyses and MD simulations, we analyze these domains as potential cholesterol binding sites and propose the possibility of multiple sterol binding pockets enabling the intramolecular transport of sterol molecules to the transmembrane domain.

Phosphorylation of a serine/proline-rich motif in oxysterol binding protein-related protein 4L (ORP4L) regulates cholesterol and vimentin binding.

The family of oxysterol binding protein (OSBP) and OSBP-related proteins (ORPs) mediate sterol and phospholipid transfer and signaling at membrane contact sites (MCS). The activity of OSBP at MCS is regulated by phosphorylation, but whether this applies to ORPs is unknown. Here we report the functional characterization of a unique proline/serine-rich phosphorylation motif (S762SPSSPSS769) in the lipid binding OSBP-related domain of full-length ORP4L and a truncated variant ORP4S. Phosphorylation was confirmed by mass spectrometry and [32P]PO4 incorporation, and in silico and in vitro assays using purified ORP4L identified putative proline-directed kinases that phosphorylate the site. The functional significance of the phospho-site was assessed by mutating serine 762, S763, S766 and S768 to aspartate or alanine to produce phosphomimetic (S4D) and phosphorylation-deficient (S4A) mutants, respectively. Solution binding of 25-hydroxycholesterol and cholesterol by recombinant ORP4L-S4D and -S4A was similar to wild-type but ORP4L-S4D more effectively extracted cholesterol from liposomes. ORP4L homo-dimerization was unaffected by phosphorylation but gel filtration of ORP4L-S4D indicated that the native conformation was affected. Confocal microscopy revealed that ORP4L-S4D also strongly associated with bundled vimentin filaments, a feature shared with ORP4S which lacks the PH and dimerization domains. We conclude that phosphorylation of a unique serine/proline motif in the ORD induces a conformation change in ORP4L that enhances interaction with vimentin and cholesterol extraction from membranes.

Influence of Cholesterol and Its Stereoisomers on Members of the Serotonin Receptor Family

Despite the ubiquity of cholesterol within the cell membrane, the mechanism by which it influences embedded proteins remains elusive. Numerous G-protein coupled receptors exhibit dramatic responses to membrane cholesterol with regard to the ligand-binding affinity and functional properties, including the 5-HT receptor family. Here, we use over 25 μs of unbiased atomistic molecular dynamics simulations to identify cholesterol interaction sites in the 5-HT1B and 5-HT2B receptors and evaluate their impact on receptor structure. Susceptibility to membrane cholesterol is shown to be subtype dependent and determined by the quality of interactions between the extracellular loops. Charged residues are essential for maintaining the arrangement of the extracellular surface in 5-HT2B; in the absence of such interactions, the extracellular surface of the 5-HT1B is malleable, populating a number of distinct conformations. Elevated cholesterol density near transmembrane helix 4 is considered to be conducive to the conformation of extracellular loop 2. Occupation of this site is also shown to be stereospecific, illustrated by differential behavior of nat-cholesterol isomers, ent- and epi-cholesterol. In simulations containing the endogenous agonist, serotonin, cholesterol binding at transmembrane helix 4 biases bound serotonin molecules toward an unexpected binding mode in the extended binding pocket. The results highlight the capability of membrane cholesterol to influence the mobility of the extracellular surface in the 5-HT1 receptor family and manipulate the architecture of the extracellular ligand-binding pocket.

Effects of high hydrostatic pressure and superfine grinding treatment on physicochemical/ functional properties of pear pomace and chemical composition of its soluble dietary fibre

In this study, we evaluated the effects of high hydrostatic pressure (HHP) and superfine grinding treatment (SFGT) on the physicochemical and functional properties of pear pomace. By using Gas chromatography and Fourier transform infrared measurement, the structural properties and monosaccharides component of dietary fibre (DF) in pear pomace were analyzed. The results showed that both HHP and SFGT could increase the soluble dietary fibre (SDF) content and change the chemical structure and monosaccharide compositions of SDF, while HHP showed a better performance in improving the main functional characteristics such as water- and oil-holding capacity, swelling and cholesterol binding capacity. Moreover, HHP treatment could effectively improve the physicochemical/functional properties of pear pomace. This study demonstrated that the pear pomace treated by HHP could be a useful fibre-rich ingredient in developing functional foods.

Synthesis and characterization of diazirine alkyne probes for the study of intracellular cholesterol trafficking

Cholesterol is an essential structural component of cellular membranes and precursor molecule for oxysterol, bile acid, and hormone synthesis. The study of intracellular cholesterol trafficking pathways has been limited in part due to a lack of suitable cholesterol analogues. Herein, we developed three novel diazirine alkyne cholesterol probes: LKM38, KK174, and KK175. We evaluated these probes as well as a previously described diazirine alkyne cholesterol analogue, trans-sterol, for their fidelity as cholesterol mimics and for study of cholesterol trafficking. LKM38 emerged as a promising cholesterol mimic because it both sustained the growth of cholesterol-auxotrophic cells and appropriately regulated key cholesterol homeostatic pathways. When presented as an ester in lipoprotein particles, LKM38 initially localized to the lysosome and subsequently trafficked to the plasma membrane and endoplasmic reticulum. LKM38 bound to diverse, established cholesterol binding proteins. Through a detailed characterization of the cellular behavior of a panel of diazirine alkyne probes using cell biological, biochemical trafficking assays and immunofluorescence approaches, we conclude that LKM38 can serve as a powerful tool for the study of cholesterol protein interactions and trafficking.