Cholesterol Binding Research Articles

Diverse Roles of Annexin A6 in Triple-Negative Breast Cancer Diagnosis, Prognosis and EGFR-Targeted Therapies.

The calcium (Ca2+)-dependent membrane-binding Annexin A6 (AnxA6), is a multifunctional, predominantly intracellular scaffolding protein, now known to play relevant roles in different cancer types through diverse, often cell-type-specific mechanisms. AnxA6 is differentially expressed in various stages/subtypes of several cancers, and its expression in certain tumor cells is also induced by a variety of pharmacological drugs. Together with the secretion of AnxA6 as a component of extracellular vesicles, this suggests that AnxA6 mediates distinct tumor progression patterns via extracellular and/or intracellular activities. Although it lacks enzymatic activity, some of the AnxA6-mediated functions involving membrane, nucleotide and cholesterol binding as well as the scaffolding of specific proteins or multifactorial protein complexes, suggest its potential utility in the diagnosis, prognosis and therapeutic strategies for various cancers. In breast cancer, the low AnxA6 expression levels in the more aggressive basal-like triple-negative breast cancer (TNBC) subtype correlate with its tumor suppressor activity and the poor overall survival of basal-like TNBC patients. In this review, we highlight the potential tumor suppressor function of AnxA6 in TNBC progression and metastasis, the relevance of AnxA6 in the diagnosis and prognosis of several cancers and discuss the concept of therapy-induced expression of AnxA6 as a novel mechanism for acquired resistance of TNBC to tyrosine kinase inhibitors.

Aster-B coordinates with Arf1 to regulate mitochondrial cholesterol transport

ObjectiveCholesterol plays a pivotal role in mitochondrial steroidogenesis, membrane structure, and respiration. Mitochondrial membranes are intrinsically low in cholesterol content and therefore must be replenished with cholesterol from other subcellular membranes. However, the molecular mechanisms underlying mitochondrial cholesterol transport remains poorly understood. The Aster-B gene encodes a cholesterol binding protein recently implicated in cholesterol trafficking from the plasma membrane to the endoplasmic reticulum (ER). In this study, we investigated the function and underlying mechanism of Aster-B in mediating mitochondrial cholesterol transport. MethodsCRISPR/Cas9 gene editing was carried out to generate cell lines deficient in Aster-B expression. The effect of Aster-B deficiency on mitochondrial cholesterol transport was examined by both confocal imaging analysis and biochemical assays. Deletion mutational analysis was also carried out to identify the function of a putative mitochondrial targeting sequence (MTS) at the N-terminus of Aster-B for its role in targeting Aster-B to mitochondria and in mediating mitochondrial cholesterol trafficking. ResultsAblation of Aster-B impaired cholesterol transport from the ER to mitochondria, leading to a significant decrease in mitochondrial cholesterol content. Aster-B is also required for mitochondrial transport of fatty acids derived from hydrolysis of cholesterol esters. A putative MTS at the N-terminus of Aster-B mediates the mitochondrial cholesterol uptake. Deletion of the MTS or ablation of Arf1 GTPase which is required for mitochondrial translocation of ER proteins prevented mitochondrial cholesterol transport, leading to mitochondrial dysfunction. ConclusionsWe identified Aster-B as a key regulator of cholesterol transport from the ER to mitochondria. Aster-B also coordinates mitochondrial cholesterol trafficking with uptake of fatty acids derived from cholesterol esters, implicating the Aster-B protein as a novel regulator of steroidogenesis.

Cholesterol Interaction with the Trimeric HIV Fusion Protein gp41 in Lipid Bilayers Investigated by Solid-State NMR Spectroscopy and Molecular Dynamics Simulations

HIV-1 entry into cells is mediated by the fusion protein gp41. Cholesterol plays an important role in this virus–cell fusion, but molecular structural information about cholesterol–gp41 interaction is so far absent. Here, we present experimental and computational data about cholesterol complexation with gp41 in lipid bilayers. We focus on the C-terminal region of the protein, which comprises a membrane-proximal external region (MPER) and the transmembrane domain (TMD). We measured peptide–cholesterol contacts in virus-mimetic lipid bilayers using solid-state NMR spectroscopy, and augmented these experimental data with all-atom molecular dynamics simulations. 2D 19F NMR spectra show correlation peaks between MPER residues and the cholesterol isooctyl tail, indicating that cholesterol is in molecular contact with the MPER–TMD trimer. 19F–13C distance measurements between the peptide and 13C-labeled cholesterol show that C17 on the D ring and C9 at the intersection of B and C rings are ~7.0 Å from the F673 side-chain 4-19F. At high peptide concentrations in the membrane, the 19F–13C distance data indicate three cholesterol molecules bound near F673 in each trimer. Mutation of a cholesterol recognition amino acid consensus motif did not change these distances, indicating that cholesterol binding does not require this sequence motif. Molecular dynamics simulations further identify two hotspots for cholesterol interactions. Taken together, these experimental data and simulations indicate that the helix-turn-helix conformation of the MPER–TMD is responsible for sequestering cholesterol. We propose that this gp41–cholesterol interaction mediates virus–cell fusion by recruiting gp41 to the boundary of the liquid-disordered and liquid-ordered phases to incur membrane curvature.

Cryo-EM structures of NPC1L1 reveal mechanisms of cholesterol transport and ezetimibe inhibition.

The intestinal absorption of cholesterol is mediated by a multipass membrane protein, Niemann-Pick C1-Like 1 (NPC1L1), the molecular target of a cholesterol lowering therapy ezetimibe. While ezetimibe gained Food and Drug Administration approval in 2002, its mechanism of action has remained unclear. Here, we present two cryo-electron microscopy structures of NPC1L1, one in its apo form and the other complexed with ezetimibe. The apo form represents an open state in which the N-terminal domain (NTD) interacts loosely with the rest of NPC1L1, leaving the NTD central cavity accessible for cholesterol loading. The ezetimibe-bound form signifies a closed state in which the NTD rotates ~60°, creating a continuous tunnel enabling cholesterol movement into the plasma membrane. Ezetimibe blocks cholesterol transport by occluding the tunnel instead of competing with cholesterol binding. These findings provide insight into the molecular mechanisms of NPC1L1-mediated cholesterol transport and ezetimibe inhibition, paving the way for more effective therapeutic development.

Peripheral myelin protein 22 preferentially partitions into ordered phase membrane domains

The ordered environment of cholesterol-rich membrane nanodomains is thought to exclude many transmembrane (TM) proteins. Nevertheless, some multispan helical transmembrane proteins have been proposed to partition into these environments. Here, giant plasma membrane vesicles (GPMVs) were employed to quantitatively show that the helical tetraspan peripheral myelin protein 22 (PMP22) exhibits a pronounced preference for, promotes the formation of, and stabilizes ordered membrane domains. Neither S-palmitoylation of PMP22 nor its putative cholesterol binding motifs are required for this preference. In contrast, Charcot-Marie-Tooth disease-causing mutations that disrupt the stability of PMP22 tertiary structure reduce or eliminate this preference in favor of the disordered phase. These studies demonstrate that the ordered phase preference of PMP22 derives from global structural features associated with the folded form of this protein, providing a glimpse at the structural factors that promote raft partitioning for multispan helical membrane proteins.

Interfacial Binding Sites for Cholesterol on Kir, Kv, K2P, and Related Potassium Channels

Inwardly rectifying, voltage-gated, two-pore domain, and related K+ channels are located in eukaryotic membranes rich in cholesterol. Here, molecular docking is used to detect specific binding sites (“hot spots”) for cholesterol on K+ channels with characteristics that match those of known cholesterol binding sites. The transmembrane surfaces of all available high-resolution structures for K+ channels were swept for potential binding sites. Cholesterol poses were found to be located largely in hollows between protein ridges. A comparison between cholesterol poses and resolved phospholipids suggests that not all cholesterol molecules binding to the transmembrane surface of a K+ channel will result in displacement of a phospholipid molecule from the surface. Competition between cholesterol binding and binding of anionic phospholipids essential for activity could explain some of the effects of cholesterol on channel function.

Intake of soluble fibre from chia seed reduces bioaccessibility of lipids, cholesterol and glucose in the dynamic gastrointestinal model simgi®

The role of soluble fibres on hypoglycemic and hypocholesterolemic effects has been widely documented, but the effect on glucose and cholesterol binding capacity of soluble fibre extracted from chia seed mucilage has not been studied until now. In the present research, dynamic gastrointestinal model simgi® combined with absorption static techniques have been used to explore the effect of chia seed mucilage at 0.75 and 0.95% w/w on the bioaccessibility of glucose, dietary lipids and cholesterol along the gastrointestinal tract.Glucose bioaccessibility was reduced when 0.95% of chia mucilage was present in sugar food models. The total reduction of glucose bioaccessibility reached a maximum of 66.7% while glucose dialysis retardation index presented its maximum of 53.4% at the end of small intestine digestion. The in vitro studies with lipid food models, showed that the presence of both, 0.75 and 0.95% of chia seed mucilage caused substantial reductions on the bioaccessibility of free fatty acids (16.8 and 56.1%), cholesterol (18.2 and 37.2% respectively) and bile salts (4.8 and 64.6%), revealing a clear dependence on fibre concentration. These innovative results highlight the potential functionality of the soluble fibre extracted from chia seeds to improve lipid and glycemic profiles and suggest the dietary health benefits of this new soluble fibre source as an ingredient in functional foods designed to reduce the risk of certain non-communicable diseases.

Soluble dietary fibers from black soybean hulls: Physical and enzymatic modification, structure, physical properties, and cholesterol binding capacity.

We used ultrasound-microwave comodification and enzyme modification (cellulase and hemicellulase) methods to extract soluble dietary fibers (SDFs) from black soybean hulls. Moreover, the structure, physical, and chemical properties, as well as the cholesterol-binding capacity of SDFs before and after modification were analyzed. The average molecular weight of SDFs extracted from raw black soybean hulls was 2.815 × 105 Da. By comparison, the average molecular weight of SDFs from ultrasound-microwave comodified hulls and enzyme-modified hulls decreased by 33.21% and 45.29%, respectively. The water-holding capacity (WHC), water-swelling capacity (WSC), and oil-holding capacity (OHC) of the extracted SDFs modified by the ultrasound-microwave method were 3.79g/g, 1.39mL/g, and 1.14g/g, respectively, a 9.54%, 23.01%, and 17.53% increase from the values of raw SDF. The WHC, WSC, and OHC of SDFs modified via the enzyme method were 3.59g/g, 1.25mL/g, and 1.03g/g, respectively, with a 3.76%, 10.62%, and 6.19% increase when compared to raw SDFs. The cholesterol-binding capacity of SDFs modified via the ultrasound-microwave and enzyme methods was 13.82 and 12.34mg/g, respectively, with an increase of 47.98% and 32.20% when compared to raw SDFs. The changes in structure and physical and chemical properties were shown to be closely related to the significantly improved cholesterol-binding capacity of the SDFs from modified black soybean hulls. This provides a theoretical basis for subsequent research and development of black soybean hulls products. PRACTICAL APPLICATION: At present, the black soybean hull, a byproduct of general grains, is usually abandoned, but black soybean hull is rich in dietary fiber. Enzymatic modification and ultrasound-microwave comodification were used to treat black soybean hull to prepare small molecular weight, highly active soluble dietary fiber. This research is of great significance to the deep processing of black soybean hull and improvement of the economic benefits of black soybean byproducts.

Probiotic Potential of Bacillus Strains Isolated from an Acidic Fermented Food Idli.

Present study is intended to assess the probiotic properties of Bacillus spp. isolated from idli batter, a traditional fermented food of Southern India and Sri Lanka. A total of 32 isolates were screened for potential pathogenic behaviour through haemolysis assay, DNase activity and antibiotics sensitivity. Two of the isolates were found to be potentially safe and identified as Bacillus spp. These strains were characterized for in vitro probiotic attributes and antioxidant activity. Both the strains showed strong acid and bile tolerance, transit tolerance, lysozyme tolerance, cell surface hydrophobicity, auto-aggregation, co-aggregation, biofilm formation potential and adhesion to human colon adenocarcinoma (HT 29) cell line demonstrating potential probiotic ability. These strains also exhibited considerable cholesterol binding, thermostability, β-galactosidase production, proteolytic, amylolytic and lipolytic activity. Cell-free supernatant inhibited the biofilm formation by Pseudomonas aeruginosa (KT266804) to 90%. Intact cells showed significant DPPH (41%), hydroxyl (31%), radical scavenging activity and lipid peroxidation inhibition (20.38%), while cell-free extracts exhibited significant superoxide anion radical scavenging activity (16.25%). Results revealed that isolates could be potential probiotic candidate after further assessment of in vivo probiotic properties and safety evaluation and could be utilised as starter cultures in functional foods.

Acute Knockdown of MG29 Alters Skeletal Muscle Cells Differentiation and Leads to Cellular Atrophy

Sarcopenia is defined as the progressive loss of skeletal muscle mass and development of force/power that occurs during aging. It is estimated that more than 30% of humans over 60 years of age suffer from sarcopenia and age‐related loss of muscle function that contributes to more than half of all chronic conditions in people over 50 and accounts for 1.5% of total U.S. healthcare expenditures (>$40 billion). Mitsugumin 29 (MG29), is a muscle‐specific protein belonging to the synaptophysin and MAL and related proteins for vesicle trafficking and membrane link (MARVEL) families of proteins which are involved in cholesterol binding and formation of oligomers of these proteins. MG29 is essential for the proper formation of the transverse‐tubule (TT) system, maintenance of lipid content of the sarcolemmal membrane, and efficient signaling between RyR1 and the store‐operated Ca2+ entry (SOCE) machinery. MG29 null (mg29−/−) mouse muscle displays abnormalities in the membrane ultrastructure of the triad junction where the TT are swollen and the sarcoplasmic reticulum (SR) networks show a vacuolated and fragmented structure. Clear phenotypic changes are seen in mg29−/− skeletal muscle at young ages (3–6 months) that resemble those seen in aged wildtype muscle. Our previous work clearly demonstrated that SOCE is compromised in an MG29‐associated manner in aged skeletal muscle and that reduced SOCE associates with a drastic decrease in MG29 content (~50%), altered intracellular Ca2+ homeostasis and decreased specific contractile force. But the function of MG29 on muscle differentiation remains unclear. In this study, we knocked down MG29 expression in mouse primary skeletal muscle cells by using MG29 siRNA. Our data shows that 48h after MG29 siRNA (10nM) transfection, the expression of MG29 decreased by ~ 80%, compared to negative control siRNA. After MG29 siRNA treatment, at day 3 of differentiation, thinner, longer myotubes were observed compared to negative controls (diameter of myotubes: negative control = 24.9 ± 2.08μm; MG29 siRNA = 19.1 ± 0.65μm, p < 0.05; Length of myotubes: negative control = 424.9 ± 50.2μm; MG29 siRNA = 580.4 ± 39.9 μm, p < 0.05). Surprisingly, fusion index (FI) was significantly increased compared to control (FI values: negative control= 53.2% ± 6.8; MG29 siRNA = 70.3% ± 7.9, p < 0.05), in agreement with increased mRNA expression of myogenenic markers MyoG and MyoD detected by real time PCR. These results suggest that the acute knock down of MG29 leads in just three days to complex myogenic changes characterized by increased myogenic differentiation but with abnormally thinner and longer muscle cells. These studies further indicate specific roles of MG29 in maintaining normal muscle mass and function, and its importance for cellular homeostasis during aging.Support or Funding InformationThis work was supported by NIH‐National Institutes of Aging R01AG056504 (NW, MB), and the George W. and Hazel M. Jay and Evanston Research Endowments (MB).

Functions of Lysosomes in Mammalian Female Reproductive System

The lysosome is the most acidic membrane-bound intracellular organelle. Lysosomal acidity is primarily maintained by vacuolar H+-ATPase (V-ATPase) and counter ion channels. There are >60 hydrolytic enzymes in the lysosome for its fundamental digestive role. Lysosomes also play important roles in endocytosis, exocytosis, autophagy, and cell death. Studies that have implicated roles of lysosomes in the female reproductive system are reviewed here. In the ovary, lysosomes are implicated in the preparation of free cholesterol for steroidogenesis and degradation of regulators of steroidogenesis, regulation of follicular atresia, follicle rupture during ovulation, luteal cell survival, and luteal regression. In the oviduct, lysosomes are involved in endocytosis of both serum and oviductal luminal components. In the uterus during the menstrual/estrous cycle, lysosomes are associated with endometrial secretion, apoptosis, and menstruation. In the uterus during early pregnancy, lysosomes are involved in the temporal and directional changes of endocytosis, uterine epithelial acidification upon embryo implantation initiation, and embryo-maternal mutual communications via extracellular vesicles. In the placenta, lysosomes are implicated in nutrient transport and placental separation from the uterus for parturition. In the mammary gland, lysosomes are important for mammary gland development and involution. These findings suggest/demonstrate functions of lysosomes in multiple processes of female reproduction, from ovulation to ovarian steroidogenesis for pregnancy maintenance, and from essential in utero nurturing of developing embryos/fetuses via embryo/fetal-maternal communications, to optional postpartum nurturing of newborns via lactation. Future studies using genetically or modified animal models and pharmacological approaches will provide novel insights into the functions and mechanisms of lysosomes in the mammalian female reproductive system.

Implication of protein glycosylation impairment in cellular cholesterol accumulation caused by Presenilin deficiency

BackgroundMajor genetic causes of familial Alzheimer disease (FAD) are mutations in genes encoding presenilin (PS) 1 and 2, respectively. PS1 and PS2 are transmembrane proteins known to constitute the catalytic subunits of the γ‐secretase complex, a membrane protease involved in proteolytic processing of amyloid precursor protein (APP) that gives rise to amyloid β‐protein (Aβ) secretion. In addiction, PSs are known to be involved in cellular cholesterol metabolism, which is fundamental for physiological cellular functions. Alterations in cholesterol metabolism could be implicated in AD development, indicated by the genetic risk factor apolipoprotein E (APOE). However, the molecular mechanisms how PSs play a role in cellular cholesterol metabolism is still not fully clarified. In this study, we characterized cholesterol metabolism in the cells deficient in either PS1 or PS2.MethodsWe have studied PS1‐ or PS2‐knock out (KO) MEFs by mass spectrometry (MS) for sterols including cholesterol and by microscopy with a cholesterol binding compound, filipin. Expression level of proteins involved in cellular cholesterol metabolism was analyzed by Western blotting (WB).ResultsCellular level of cholesterol as well as its precursors, desmosterol and lathosterol, were elevated in PS1‐ and PS2‐KO as compared to WT MEFs. Fluorescence microscopy also revealed intracellular cholesterol accumulation in PS1 and PS2 KO cells. Interestingly, the subcellular distribution of cholesterol was different between PS1‐ and PS2‐KO MEFs. We further analyzed expression of proteins involved in cellular cholesterol metabolism, including NPC1, an intracellular cholesterol transporter whose deficiency leads to lysosomal cholesterol storage and neurodegeneration in Niemann‐Pick disease type C. The deficiency of PS1‐ and PS2‐KO MEFs was associated with altered glycosylation of NPC1. The glycosylation and expression of other membrane proteins, such as N‐cadherin, LAMP2, and LRP1 was also dependent on the PS genotype. To investigate the role of protein glycosylation in cellular cholesterol metabolism, we chronically treated wild‐type MEFs with several inhibitors of protein glycosylation targeting different steps of glycosylation processes. The inhibitors differentially affected protein glycosylation pattern and subcellular cholesterol distribution. Importantly, some of the inhibitors partially mimicked the cellular cholesterol accumulation as well as the protein glycosylation pattern observed in PS1‐ and PS2‐KO MEFs.Discussion/ConclusionsThese results suggest that deficiency of PS1 or PS2 differentially affect cellular cholesterol metabolism via impairment of protein glycolsylation. The exact mechanisms of altered expression of NPC1 as well as γ‐secretase activity in the impairment of cholesterol metabolism and protein glycosylation remain to be elucidated. It will be interesting to further dissect the role of protein glycosylation and cholesterol mismetabolism in the pathogenesis of AD.Support or Funding InformationGerman Research Foundation (DFG) (Project number: 348207315, Naoto Oikawa)

Structural Similarity with Cholesterol Reveals Crucial Insights into Mechanisms Sustaining the Immunomodulatory Activity of the Mycotoxin Alternariol.

The proliferation of molds in domestic environments can lead to uncontrolled continuous exposure to mycotoxins. Even if not immediately symptomatic, this may result in chronic effects, such as, for instance, immunosuppression or allergenic promotion. Alternariol (AOH) is one of the most abundant mycotoxins produced by Alternaria alternata fungi, proliferating among others in fridges, as well as in humid walls. AOH was previously reported to have immunomodulatory potential. However, molecular mechanisms sustaining this effect remained elusive. In differentiated THP-1 macrophages, AOH hardly altered the secretion of pro-inflammatory mediators when co-incubated with lipopolysaccharide (LPS), opening up the possibility that the immunosuppressive potential of the toxin could be related to an alteration of a downstream pro-inflammatory signaling cascade. Intriguingly, the mycotoxin affected the membrane fluidity in macrophages and it synergistically reacted with the cholesterol binding agent MβCD. In silico modelling revealed the potential of the mycotoxin to intercalate in cholesterol-rich membrane domains, like caveolae, and immunofluorescence showed the modified interplay of caveolin-1 with Toll-like Receptor (TLR) 4. In conclusion, we identified the structural similarity with cholesterol as one of the key determinants of the immunomodulatory potential of AOH.

Lipid-Protein Interactions Are a Unique Property and Defining Feature of G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) are membrane-bound proteins that depend on their lipid environment to carry out their physiological function. Combined efforts from many theoretical and experimental studies on the lipid-protein interaction profile of several GPCRs hint at an intricate relationship of these receptors with their surrounding membrane environment, with several lipids emerging as particularly important. Using coarse-grained molecular dynamics simulations, we explore the lipid-protein interaction profiles of 28 different GPCRs, spanning different levels of classification and conformational states and totaling to 1 ms of simulation time. We find a close relationship with lipids for all GPCRs simulated, in particular, cholesterol and phosphatidylinositol phosphate (PIP) lipids, but the number, location, and estimated strength of these interactions is dependent on the specific GPCR as well as its conformational state. Although both cholesterol and PIP lipids bind specifically to GPCRs, they utilize distinct mechanisms. Interactions with PIP lipids are mediated by charge-charge interactions with intracellular loop residues and stabilized by one or both of the transmembrane helices linked by the loop. Interactions with cholesterol, on the other hand, are mediated by a hydrophobic environment, usually made up of residues from more than one helix, capable of accommodating its ring structure and stabilized by interactions with aromatic and charged/polar residues. Cholesterol binding to GPCRs occurs in a small number of sites, some of which (like the binding site on the extracellular side of transmembrane 6/7) are shared among many class A GPCRs. Combined with a thorough investigation of the local membrane structure, our results provide a detailed picture of GPCR-lipid interactions. Additionally, we provide an accompanying website to interactively explore the lipid-protein interaction profile of all GPCRs simulated to facilitate analysis and comparison of our data.

NPC1b as a novel target in controlling the cotton bollworm, Helicoverpa armigera.

Insects cannot synthesize sterols and must acquire them from food. The mechanisms underlying how insects uptake dietary sterols are largely unknown except that NPC1b, an integral membrane protein, has been shown to be responsible for dietary cholesterol uptake in Drosophila melanogaster. However, whether NPC1b orthologs in other insect species, particularly the economically important pests, function similarly remains to be determined. In this study, we characterized the function of NPC1b in Helicoverpa armigera, a global pest that causes severe yield losses to many important crops. Limiting dietary cholesterol uptake to insects significantly inhibited food ingestion and weight gain. Compared to the wild-type H. armigera, the CRISPR/Cas9-edited NPC1b mutant larvae were incapable of getting adequate cholesterol and died in their early life stage. Gene expression profile and in situ hybridization analyses indicated that NPC1b was mainly expressed in the midgut where dietary cholesterol was absorbed. Expression of NPC1b was also correlated with the feeding life stages and was especially upregulated during early larval instars. Protein-ligand docking and sequence similarity analyses further demonstrated that NPC1b proteins of lepidopteran insects shared a relatively conserved cholesterol binding region, NPC1b_NTD, which, however, was highly divergent from bees-derived sequences. NPC1b was crucial for dietary cholesterol uptake and growth of H. armigera, and therefore could serve as an insecticide target for the development of a novel pest-management approach to control this economically significant insect pest with little off-target effect on bees and sterol-autotrophic animals. © 2020 Society of Chemical Industry.

Modification of insoluble dietary fiber from ginger residue through enzymatic treatments to improve its bioactive properties

To add value ginger residues containing insoluble dietary fiber (IDF), multiple enzymes were used to obtain modified insoluble dietary fiber (MIDF). The water retention capacity (WRC), water swelling capacity (WSC) and oil binding capacity (OBC) for lard of MIDF were 22.45%, 33.85% and 36.26% higher than those of IDF, respectively. Meanwhile, MIDF showed higher cation exchange capacity (CEC) than that of IDF. Cholesterol, sodium cholate and sodium nitrite binding capacity (CBC, SCBC, SNBC) of MIDF were 70.28%, 42.07% and 15.9% higher than those of IDF at pH 7, respectively. And CBC and SNBC were 21.18% and 12.17% higher than those of IDF at pH 2, respectively. Compared to IDF, MIDF exposed more hydrophilic groups and more specific surface areas. The oral administration showed that MIDF significantly reduced total cholesterol (TC), triglyceride (TG) and atherogenic index (AI) by 12.23%, 25.15% and 60.63%, respectively (P < 0.05), but significantly increased the high density lipoprotein-cholesterol (HDL-C) by 32.93% to the normal level (P < 0.05). Furthermore, histopathology of liver also indicated that MIDF could reduce the risk of fatty liver. These properties demonstrated that MIDF showed better physicochemical and functional properties than IDF. Thus MIDF would be a potentially available food ingredient in weight-loss functional food processing.

The Molecular Mechanisms of Cholesterol Regulation of Kir Channels Revealed by Direct and Quantitative Approaches

Inwardly rectifying potassium (Kir) channels regulate cell excitability in multiple tissues and are key modulators of cardiac and vascular electrical activity and contractility. Channel activity is regulated by various ligands including membrane lipids. Phosphoinositol-4,5-bisphosphate (PIP2) is the absolutely required agonist for all Kir channel subfamily members while cholesterol exhibits mixed effects depending on the subfamily; it inhibits Kir2 but increases the open probability of Kir3 channels. In order to unambiguously determine cholesterol binding sites as well as regulatory mechanisms, quantitative and more direct approaches have been developed. First, an in vitro functional assay system has been established to measure Kir channel activity in the presence of cholesterol as high as 40 mol%. Second, photocrosslinkable cholesterol mimetics (LKM38) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) approaches have been developed to directly detect channel residues in close proximity to cholesterol within membranes. With these tools, we demonstrate both concentration-dependent and non-competitive Kir2 channel inhibition by cholesterol, suggesting that cholesterol has binding site(s) not affecting PIP2 binding to the channel. Further, we detect two cholesterol-interacting sites in MS experiments, one previously identified[1], and a novel site that has not been suggested. The same approaches will be repeated for Kir3 channels and subsequent docking and molecular dynamics simulations may enlighten the molecular mechanisms of the differential cholesterol regulations of Kir channels. 1. Rosenhouse-Dantsker, A., et al., Identification of novel cholesterol-binding regions in Kir2 channels. J Biol Chem, 2013. 288(43): p. 31154-64.

Structural Determinants of Cholesterol Recognition in Helical Membrane Proteins

Cholesterol is an integral component of mammalian membranes. It has been shown to modulate membrane dynamics and alter protein function. However, understanding the molecular mechanisms of how cholesterol alters protein function through specific interactions is complicated by our inability to differentiate cholesterol as a ligand and as a lipid in the membrane. Specifically, it is difficult to discern if a cholesterol-membrane protein interaction observed in an experimental structure is specific, unspecific, or a crystallization artefact. The only widely recognized search algorithm for cholesterol binding pockets is sequence-based, i.e. the CRAC and CARC motifs. While, both motifs have been identified on multiple proteins, there is inconclusive evidence on the necessity and sufficiency of its role in identifying cholesterol functional modifiers on its own. To elucidate cholesterol's role on the functional changes of an array of protein types, we characterized the rate of evolution of interacting residues, binding pocket residue types, and binding pocket spatial arrangement of high-resolution crystal structures of cholesterol-membrane protein complexes in the protein data bank (PDB). We compare the predictive power of this multi-layer analysis to the outcomes of the sequence-based CRAC and CARC motifs. Information gleaned from our characterization will eventually enable a structure-based approach for prediction and design of cholesterol binding sites.

Lipid accumulation product and late-onset hypogonadism in middle-aged and elderly men: results from a cross-sectional study in China

ObjectivesHypogonadism in men is related to the deterioration of general health. However, the association between lipid overaccumulation and ageing-related hypogonadism remains an undetermined concept. We aimed to provide an insight...

Use of Giant Plasma Membrane Vesicles (GPMV) to Examine the Lo/Ld Phase Preference of the C99 Domain of the Amyloid Precursor Protein

Alzheimer's disease (AD) is the most common form of dementia at the moment cannot be treated, prevented, or even delayed. Despite decades of intensive research efforts, there are still many mysteries regarding the etiology of AD. The predominant hypothesis for AD is that disease pathology begins decades before clinically-detectable symptoms due to the formation of toxic oligomers and aggregates composed mainly of amyloid-β (Aβ) peptides. Amyloid-β is generated when the amyloid precursor protein (APP) transmembrane C-terminal fragment, C99, is cleaved in the membrane by γ-secretase. Cholesterol has long been implicated in AD and shown to increase the generation of Aβ peptides, but it is not known how cholesterol or cholesterol-rich environments impact C99 membrane localization. We are examining the membrane phase preference of C99 localization in membranes using giant plasma membrane-derived vesicles (GPMVs). These cell-derived giant vesicles exhibit phase separation into cholesterol-rich Lo and Ld-like domains that can be directly observed using confocal microscopy. We compare and contrast various methods for preparing GPMVs and report the phase partitioning behavior of both WT C99 and cholesterol binding site-disrupted mutant forms.