Structural insight into GPR155-mediated cholesterol sensing and signal transduction.

Intrinsically disorderedproteins (IDPs) are implicated in manyhuman diseases and are increasingly being pursued as drug targets.Conventional structure-based drug design methods that rely on well-definedbinding sites are, however, largely unsuitable for IDPs. Here, wepresent computationally efficient ensemble docking approaches to predictthe relative affinities of small molecules to IDPs and characterizetheir dynamic, heterogeneous binding mechanisms at atomic resolution.We show that these ensemble docking protocols accurately predict therelative binding affinities of three small molecule α-synucleinligands measured by NMR spectroscopy and generate conformational ensemblesof ligand binding modes in remarkable agreement with experimentallyvalidated long-time scale molecular dynamics simulations of ligandbinding. Our results demonstrate the potential of ensemble dockingapproaches for predicting small molecule binding to IDPs and suggestthat these methods may be valuable tools for IDP drug discovery campaigns.

Long-time scale molecular dynamics study of Co diffusion on the Au([formula omitted]) surface

The effect of dietary cholesterol on the composition of human bile.

This study was designed to investigate the effects of pectin on cholesterol and bile acid metabolism and to elucidate the mechanisms involved in its hypolipidemic effect in rats. The key regulatory enzymes in cholesterol and bile acid metabolism, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and cholesterol 7α-hydroxylase, were determined. Circulating, hepatic, and biliary lipid concentrations and fecal bile acid excretion were also measured. Male Wistar rats were fed a fiber-free or a pectin-supplemented (7 g/100 g) diet for 4 wk. Bile flow and the biliary secretion of both bile acids and cholesterol were not significantly different than controls in pectin-fed rats. The addition of pectin to the diet resulted in lower serum and liver cholesterol concentrations (-27 and -17%, respectively). Fecal bile acid excretion (+168%) and the hepatic activity of cholesterol 7α-hydroxylase (+70%) were significantly higher in pectin-fed animals. HMG-CoA reductase activity was also significantly greater (+11%) in the presence of dietary pectin. Results of our study indicate that pectin, by enhancing fecal bile acid excretion, may cause increased hepatic synthesis of bile acids and liver depletion of cholesterol in rats, which results in a higher rate of cholesterol synthesis and reduced serum cholesterol concentrations.

Dysregulated autophagy contributes to liver steatosis, yet its regulation under distinct metabolic contexts remains poorly defined. Here, we identify bile acids (BAs) as critical modulators of hepatic autophagy. Circulating BA levels are elevated in human subjects with liver steatosis and independently associated with increased hepatic steatosis risk. High-fat diet (HFD) feeding increases circulating BA levels, while simultaneously reducing hepatic autophagic flux in mice, whereas pharmacological inhibition of farnesoid X receptor (FXR) enhances autophagy and alleviates steatosis in the livers of HFD-fed mice. Mechanistically, circulating BAs promote hepatic acetyl-CoA production through FXR-induced acyl-CoA oxidase 1 (ACOX1), which in turn suppresses autophagy by increasing the mechanistic target of rapamycin complex 1 (mTORC1) signaling. Similar to HFD feeding, prolonged fasting elevates BA levels and hepatic lipid accumulation, while concurrently upregulating hepatic miR-378, a positive regulator of BA synthesis. Although miR-378 exerts a cell-autonomous pro-autophagic effect during short-term fasting, it paradoxically drives lipid accumulation by suppressing hepatic autophagy via BA/FXR/ACOX1/acetyl-CoA axis in a non-cell-autonomous manner during either HFD feeding or prolonged fasting when BA action becomes considerable. Together, our study uncovers BAs as a previously unrecognized class of inhibitors of hepatic autophagy during prolonged fasting and in metabolic dysfunction-associated steatotic liver disease (MASLD), providing novel insights into context-dependent autophagic regulation of hepatic lipid metabolism and potential therapeutic strategies for MASLD.

Proteins typically adopt a multitude of flexible and rapidly interconverting conformers, many of which are governed by specific protein interaction domains. Whereas disc-shaped oligomeric HDL and its major protein component ApoA1 have been the focus of several investigations, the structural properties of monomeric ApoA1 remain poorly understood. Using tens of independent molecular simulations (>50 μs), we reveal that ApoA1 adopts a compact conformation. Upon the addition of a physiological concentration of cholesterol to ApoA1, the monomeric protein spontaneously formed a circular conformation. Remarkably, these drastic structural perturbations are driven by a specific cholesterol binding site at the C-terminal and a novel cholesterol binding site at the N-terminal. We propose a mechanism whereby ApoA1 opens in a stagewise manner and mutating the N-terminal binding site destroys the open "belt-shaped" topology. Complementary experiments confirm that the structural changes are induced by specific association of cholesterol with ApoA1, not by the nonspecific hydrophobic effect.

Der p 2 is a major dust mite allergen and >80% of mite allergic individuals have specific IgE to this allergen. Although it is well characterized in terms of allergenicity, there is still some ambiguity in terms of its biological function. Three-dimensional structural analysis of Der p 2 and its close homologues indicate the presence of a hydrophobic cavity which can potentially bind to lipid molecules. In this study, we aimed to identify the potential ligand of Der p 2. Using a liposome pulldown assay, we show that recombinant Der p 2 binds to liposomes prepared with exogenous cholesterol in a dose dependent fashion. Next, an ELISA based assay using immobilized lipids was used to study binding specificities of other lipid molecules. Cholesterol was the preferred ligand of Der p 2 among 11 different lipids tested. Two homologues of Der p 2, Der f 2 and Der f 22 also bound to cholesterol. Further, using liquid chromatography-mass spectrometry (LC-MS), we confirmed that cholesterol is the natural ligand of Der p 2. Three amino acid residues of Der p 2, V104, V106 and V110 are possible cholesterol binding sites, as alanine mutations of these residues showed a significant decrease in binding (p < 0.05) compared to wild-type Der p 2. These results provide the first direct experimental evidence that Der p 2 binds to cholesterol.

Interactive effects of dietary cholesterol and bile acids on the growth, lipid metabolism, immune response and intestinal microbiota of Litopenaeus vannamei: Sparing effect of bile acids on cholesterol in shrimp diets

Objective To explore the potential mechanisms of low density lipoprotein receptor (LDLr) in high glucose peritoneal dialysis solution (PDS)-induced peritoneal fibrosis. Methods Human peritoneal mesothelial cells (PMCs) were applied. In pre-experiment, human PMCs were cultured with 1.5% PDS, 2.5% PDS and 4.25% PDS for 6 h, 12 h and 24 h. 4.25% mannitol was used as high osmotic pressure control. In formal experiment, PMCs were divided into the control group(treated with phosphate buffer saline) and the high glucose PDS group (treated with 4.25% PDS for 24 h). Morphological change of PMCs was observed by inverted microscope. The mRNA and protein expressions of extracellular matrix proteins such as α-smooth muscle actin (α-SMA), fibroblast specific protein-1 (FSP-1) and collagenⅠ in PMCs were respectively measured by real-time PCR and Western blotting. The lipid accumulation was observed by oil red O staining and filipin staining, and the content of intracellular cholesterol ester was detected by high-performance liquid chromatography. The co-expression of the sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) with golgin was observed with immunofluorescent staining. The mRNA and protein expressions of LDLr, SREBP-2 and SCAP were respectively detected by real-time PCR and Western blotting. The mRNA and protein expressions of mammalian target of rapamycin (mTOR), eukaryotic initiation factor 4E-binding protein 1 (4EBP1), and p70 S6 kinase (S6K1) were respectively detected by real-time PCR and Western blotting. Results (1) Compared with the 1.50% PDS stimulation, 4.25% PDS for 24 h intervention significantly increased the expression of LDLr in PMCs (P 0.05). (2) Compared with those in the control group, in high glucose PDS group PMCs showed notable elongation consistent with the morphology of myofibroblasts, the expressions of α-SMA, FSP-1 and collagen Ⅰ were increased (all P<0.05), and the intracellular cholesterol were enhanced (P<0.05). Meanwhile, the co-expression of SCAP with golgin was enhanced, and the mRNA and protein expressions of LDLr, SREBP-2 and SCAP were up-regulated in high glucose PDS group (all P<0.05). Further, the mRNA and protein phosphorylation of mTOR, 4EBP1 and S6K1 were increased (all P<0.05). Conclusions The disruption of LDLr feedback regulation is involved in high glucose PDS-mediated cholesterol accumulation in PMCs by mammalian target of rapamycin complex 1 (mTORC1) pathway, which promotes the accumulation of extracellular matrix and peritoneal fibrosis. Key words: Peritoneal dialysis; Receptors, LDL; Intracellular signaling peptides and proteins; Retroperitoneal fibrosis; Mechanistic target of rapamycin complex 1

Bile acids are implicated as aetiological factors in many types of gastrointestinal tract cancer including cholangiocarcinoma (CCA). Alterations in bile acid concentrations may affect the pathogenesis of these different types of cancer. Our aim was to determine the bile acid profile in gallbladder bile from patients who underwent liver resection. Thirty-seven patients with cholangiocarcinoma, 5 with hepatocellular carcinoma, and 7 with benign biliary diseases were studied. High pressure liquid chromatography was used to analyze conjugated and unconjugated bile acids. CCA patients with low (≤ 2 mg/dl) and high (>2 mg/dl) levels of total serum bilirubin had significantly higher total bile acid and conjugated bile acid concentrations than the benign biliary disease group. Markedly elevated levels of cholic and chenodeoxycholic acid were found in CCA cases with high levels of total serum bilirubin. Concentrations of total bile acids and primary bile acid were correlated with serum cholesterol, bilirubin and ALP in CCA. Notably, correlation of the carcinoembryonic antigen, a tumor marker, was found with level of total bile acids and chenodeoxycholic acid. These findings suggest a different pattern of bile acid concentration in cancer patients compared to patients with benign biliary diseases. Thus, accumulation of certain bile acids may be involved in carcinogenesis.

Interfacial binding sites for cholesterol on GABAA receptors and competition with neurosteroids

The cytokine interferon‐gamma (IFN‐γ) is a master regulator of innate and adaptive immunity involved in a broad array of human diseases that range from atherosclerosis to cancer. IFN‐γ exerts it signaling action by binding to a specific cell surface receptor, the IFN‐γ receptor (IFN‐γR), whose activation critically depends on its partition into lipid nanodomains. However, little is known about the impact of specific lipids on IFN‐γR signal transduction activity. Here, a new conserved cholesterol (chol) binding motif localized within its single transmembrane domain is identified. Through direct binding, chol drives the partition of IFN‐γR2 chains into plasma membrane lipid nanodomains, orchestrating IFN‐γR oligomerization and transmembrane signaling. Bioinformatics studies show that the signature sequence stands for a conserved chol‐binding motif presented in many mammalian membrane proteins. The discovery of chol as the molecular switch governing IFN‐γR transmembrane signaling represents a significant advance for understanding the mechanism of lipid selectivity by membrane proteins, but also for figuring out the role of lipids in modulating cell surface receptor function. Finally, this study suggests that inhibition of the chol‐IFNγR2 interaction may represent a potential therapeutic strategy for various IFN‐γ‐dependent diseases.

This work evaluates lactic acid bacteria (LAB) obtained from fermented mustards for lowering cholesterol. The LAB strains’ ability to reduce cholesterol in vitro was determined using activity of bile salt hydrolase (BSH) and deconjugation of bile salt in vitro. The LAB strains were examined for their ability to resist acid and bile salt. Strains that can reduce cholesterol adhere to Caco-2 cells and they were identified. 9 LAB isolates had higher total BSH activity (1.07 to 1.92 U/mL) than the reference strains. BSH specific activity range from 1.33 to 3.13 U/mg. Due to different protein contents in the cell extracts, there was no good correlation between the exact BSH activity and total BSH activity by most of the isolates. All of the isolates deconjugated sodium glycocholate, freeing cholic acid, from 1.02 to 2.64 mM. The tested isolates deconjugated sodium glycocholate at different levels. All tested strains exhibited acid tolerance and bile tolerance; they can survive well at pH 3.0 for 3 h and bile tolerance for 24 h. Adhesion of the tested strains to the Caco-2 cell lines, strain B0007 and B0008 strongly adhere in comparison to the control strains and other isolated strains. These results demonstrated positive effect of isolates on BSH activity, deconjugation and thereby leading to the probiotic potential of lowering cholesterol. Key words: Lactic acid bacteria, acid- and bile-tolerant, bile salt hydrolase activity, deconjugation, adhesion activity.

Lipoprotein metabolism in FIC1 deficiency due to ATP8B1 mutations has never been studied sufficiently. This study was performed to investigate the detailed lipoprotein metabolism in benign recurrent intrahepatic cholestasis (BRIC) caused by FIC1 deficiency. Lipoprotein profile and major lipoprotein regulators such as lecithin:cholesterol acyltransferase (LCAT), hepatic triglyceride lipase (HTGL), lipoprotein lipase, and cholesteryl ester transfer protein in a Japanese patient with BRIC were serially examined during a bout of cholestasis. Liver expression of farnesoid X receptor (FXR), which suppresses high-density lipoprotein (HDL) generation, was also examined. Hypercholesterolemia and lipoprotein X accumulation were never observed throughout this study. When the cholestasis was severe, triglyceride-rich low-density lipoprotein (LDL) accounted for most of the plasma lipoproteins whereas HDL was hardly detectable. Concurrently, activities of all regulators were decreased, together with decreases of the serum parameter for liver protein synthesis. In particular, suppressions of LCAT and HTGL activities were severe and greatly contributed to the appearance of triglyceride-rich LDL. As the cholestasis improved, this LDL gradually transformed into normal LDL with the recoveries of LCAT and HTGL activities. The activities of all regulators for the last 1 to 2 months were normal but HDL remained depleted. His liver showed low FXR expression compared with control livers. The present study showed an appearance of triglyceride-rich LDL due to suppressions of LCAT and HTGL activities and a depletion of HDL that is not able to be explained by lipoprotein regulators or FXR in our patient.

Cholesterol–bile salt vesicles as potential delivery vehicles for drug and vaccine delivery