Cholesterol Binding Sites Research Articles

Structural basis of frizzled 7 activation and allosteric regulation

Frizzleds (ten paralogs: FZD1-10) belong to the class F of G protein-coupled receptors (GPCRs), which remains poorly understood despite its crucial role in multiple key biological functions including embryonic development, stem cell regulation, and homeostasis in the adult. FZD7, one of the most studied members of the family, is more specifically involved in the migration of mesendoderm cells during the development and renewal of intestinal stem cells in adults. Moreover, FZD7 has been highlighted for its involvement in tumor development predominantly in the gastrointestinal tract. This study reports the structure of inactive FZD7, without any stabilizing mutations, determined by cryo-electron microscopy (cryo-EM) at 1.9 Å resolution. We characterize a fluctuating water pocket in the core of the receptor important for FZD7 dynamics. Molecular dynamics simulations are used to investigate the temporal distribution of those water molecules and their importance for potential conformational changes in FZD7. Moreover, we identify lipids interacting with the receptor core and a conserved cholesterol-binding site, which displays a key role in FZD7 association with a transducer protein, Disheveled (DVL), and initiation of downstream signaling and signalosome formation.

Cholesterol Allosterically Modulates the Structure and Dynamics of the Taurocholate Export Pump (ABCB11).

The BSEP/ABCB11 transmembrane protein translocates taurine- and glycine-conjugated bile salts across the hepatocyte bilayer driven by ATP-hydrolysis. Direct inhibition of BSEP/ABCB11 leads to idiosyncratic drug-induced liver injury. ABCB11 is localized within the cholesterol-enriched lipid raft, and membrane cholesterol depletion leads to impaired taurocholate transport. However, structural insight into the mechanism of the cholesterol-mediated regulation of ABCB11 activity remains elusive. We used extensive molecular dynamics simulation coupled with well-tempered metadynamics to elucidate the role of membrane cholesterol in the structure and dynamics of ABCB11. We identified specific high-residence binding sites for cholesterol within the transmembrane domain. The free-energy simulations have elucidated that the bound cholesterol stabilizes the "inward-open" conformation of the protein. Cholesterol-ABCB11 interactions trigger allosteric communications between the transmembrane and nucleotide-binding domains through the linker region. Cholesterol depletion destabilizes the allosteric network of the protein. As a result, it adopts a more collapsed form with a reduced volume of the taurocholate-binding pocket.

Improved Highly Mobile Membrane Mimetic Model for Investigating Protein-Cholesterol Interactions.

Cholesterol (CHL) plays an integral role in modulating the function and activity of various mammalian membrane proteins. Due to the slow dynamics of lipids, conventional computational studies of protein-CHL interactions rely on either long-time scale atomistic simulations or coarse-grained approximations to sample the process. A highly mobile membrane mimetic (HMMM) has been developed to enhance lipid diffusion and thus used to facilitate the investigation of lipid interactions with peripheral membrane proteins and, with customized in silico solvents to replace phospholipid tails, with integral membrane proteins. Here, we report an updated HMMM model that is able to include CHL, a nonphospholipid component of the membrane, henceforth called HMMM-CHL. To this end, we had to optimize the effect of the customized solvents on CHL behavior in the membrane. Furthermore, the new solvent is compatible with simulations using force-based switching protocols. In the HMMM-CHL, both improved CHL dynamics and accelerated lipid diffusion are integrated. To test the updated model, we have applied it to the characterization of protein-CHL interactions in two membrane protein systems, the human β2-adrenergic receptor (β2AR) and the mitochondrial voltage-dependent anion channel 1 (VDAC-1). Our HMMM-CHL simulations successfully identified CHL binding sites and captured detailed CHL interactions in excellent consistency with experimental data as well as other simulation results, indicating the utility of the improved model in applications where an enhanced sampling of protein-CHL interactions is desired.

Free-Standing DNA Origami Superlattice to Facilitate Cryo-EM Visualization of Membrane Vesicles.

Technological breakthroughs in cryo-electron microscopy (cryo-EM) methods open new perspectives for highly detailed structural characterizations of extracellular vesicles (EVs) and synthetic liposome-protein assemblies. Structural characterizations of these vesicles in solution under a nearly native hydrated state are of great importance to decipher cell-to-cell communication and to improve EVs' application as markers in diagnosis and as drug carriers in disease therapy. However, difficulties in preparing holey carbon cryo-EM grids with low vesicle heterogeneities, at low concentration and with kinetic control of the chemical reactions or assembly processes, have limited cryo-EM use in the EV study. We report a straightforward membrane vesicle cryo-EM sample preparation method that assists in circumventing these limitations by using a free-standing DNA-affinity superlattice for covering holey carbon cryo-EM grids. Our approach uses DNA origami to self-assemble to a solution-stable and micrometer-sized ordered molecular template in which structure and functional properties can be rationally controlled. We engineered the template with cholesterol-binding sites to specifically trap membrane vesicles. The advantages of this DNA-cholesterol-affinity lattice (DCAL) include (1) local enrichment of artificial and biological vesicles at low concentration and (2) isolation of heterogeneous cell-derived membrane vesicles (exosomes) from a prepurified pellet of cell culture conditioned medium on the grid.

Dual pattern of cholesterol-induced decoupling of residue-residue interactions of Kir2.2

Cholesterol is a negative regulator of a variety of ion channels. We have previously shown that cholesterol suppresses Kir2.2 channels via residue-residue uncoupling on the inter-subunit interfaces within the close state of the channels (3JYC). In this study, we extend this analysis to the other known structure of Kir2.2 that is closer to the open state of Kir2.2 channels (3SPI) and provide additional analysis of the residue distances between the uncoupled residues and cholesterol binding domains in the two conformation states of the channels. We found that the general phenomenon of cholesterol binding leading to uncoupling between specific residues is conserved in both channel states but the specific pattern of the uncoupling residues is distinct between the two states and implies different mechanisms. Specifically, we found that cholesterol binding in the 3SPI state results in an uncoupling of residues in three distinct regions; the transmembrane domain, membrane-cytosolic interface, and the cytosolic domain, with the first two regions forming an envelope around PI(4,5)P2 and cholesterol binding sites and the distal region overlapping with the subunit-subunit interface characterized in our previous study of the disengaged state. We also found that this uncoupling is dependent upon the number of cholesterol molecules bound to the channel. We further generated a mutant channel Kir2.2P187V with a single point mutation in a residue proximal to the PI(4,5)P2 binding site, which is predicted to be uncoupled from other residues in its vicinity upon cholesterol binding and found that this mutation abrogates the sensitivity of Kir2.2 to cholesterol changes in the membrane. These findings suggest that cholesterol binding to this conformation state of Kir2.2 channels may destabilize the PI(4,5)P2 interactions with the channels while in the disengaged state the destabilization occurs where the subunits interact. These findings give insight into the structural mechanistic basis for the functional effects of cholesterol binding to the Kir2.2 channel.

Comprehensive Characterization of LAT1 Cholesterol-Binding Sites.

The human L-type amino acid transporter 1 (LAT1; SLC7A5), is an amino acid exchanger protein, primarily found in the blood-brain barrier, placenta, and testis, where it plays a key role in amino acid homeostasis. Cholesterol is an essential lipid that has been highlighted to play a role in regulating the activity of membrane transporters, such as LAT1, yet little is known about the molecular mechanisms driving this phenomenon. Here we perform a comprehensive computational analysis to investigate cholesterol's role in LAT1 structure and function, focusing on four cholesterol-binding sites (CHOL1-4) identified in a recent LAT1-apo inward-open conformation cryo-EM structure. Through a series of independent molecular dynamics (MD) simulations, molecular docking, MM/GBSA free energy calculations, and other analysis tools, we explored the interactions between LAT1 and cholesterol. Our findings suggest that CHOL3 forms the most stable and favorable interactions with LAT1. Principal component analysis (PCA) and center of mass (COM) distance assessments show that CHOL3 binding stabilizes the inward-open state of LAT1 by preserving the spatial arrangement of the hash and bundle domains. Additionally, we propose an alternative cholesterol-binding site for originally assigned CHOL1. Overall, this study improves the understanding of cholesterol's modulatory effect on LAT1 and proposes candidate sites for the discovery of future allosteric ligands with rational design.

State-dependent binding of cholesterol and an anionic lipid to the muscle-type Torpedo nicotinic acetylcholine receptor

The ability of the Torpedo nicotinic acetylcholine receptor (nAChR) to undergo agonist-induced conformational transitions requires the presence of cholesterol and/or anionic lipids. Here we use recently solved structures along with multiscale molecular dynamics simulations to examine lipid binding to the nAChR in bilayers that have defined effects on nAChR function. We examine how phosphatidic acid and cholesterol, lipids that support conformational transitions, individually compete for binding with phosphatidylcholine, a lipid that does not. We also examine how the two lipids work synergistically to stabilize an agonist-responsive nAChR. We identify rapidly exchanging lipid binding sites, including both phospholipid sites with a high affinity for phosphatidic acid and promiscuous cholesterol binding sites in the grooves between adjacent transmembrane α-helices. A high affinity cholesterol site is confirmed in the inner leaflet framed by a key tryptophan residue on the MX α-helix. Our data provide insight into the dynamic nature of lipid-nAChR interactions and set the stage for a detailed understanding of the mechanisms by which lipids facilitate nAChR function at the neuromuscular junction.

Interaction with stomatin directs human proton channels into cholesterol-dependent membrane domains

Many membrane proteins are modulated by cholesterol. Here we report profound effects of cholesterol depletion and restoration on the human voltage-gated proton channel, hHV1, in excised patches but negligible effects in the whole-cell configuration. Despite the presence of a putative cholesterol-binding site, a CARC motif in hHV1, mutation of this motif did not affect cholesterol effects. The murine HV1 lacks a CARC sequence but displays similar cholesterol effects. These results argue against a direct effect of cholesterol on the HV1 protein. However, the data are fully explainable if HV1 preferentially associates with cholesterol-dependent lipid domains, or “rafts.” The rafts would be expected to concentrate in the membrane/glass interface and to be depleted from the electrically accessible patch membrane. This idea is supported by evidence that HV1 channels can diffuse between seal and patch membranes when suction is applied. Simultaneous truncation of the large intracellular N and C termini of hHV1 greatly attenuated the cholesterol effect, but C truncation alone did not; this suggests that the N terminus is the region of attachment to lipid domains. Searching for abundant raft-associated proteins led to stomatin. Co-immunoprecipitation experiment results were consistent with hHV1 binding to stomatin. The stomatin-mediated association of HV1 with cholesterol-dependent lipid domains provides a mechanism for cells to direct HV1 to subcellular locations where it is needed, such as the phagosome in leukocytes.

A cholesterol-binding bacterial toxin provides a strategy for identifying a specific Scap inhibitor that blocks lipid synthesis in animal cells

Lipid synthesis is regulated by the actions of Scap, a polytopic membrane protein that binds cholesterol in membranes of the endoplasmic reticulum (ER). When ER cholesterol levels are low, Scap activates SREBPs, transcription factors that upregulate genes for synthesis of cholesterol, fatty acids, and triglycerides. When ER cholesterol levels rise, the sterol binds to Scap, triggering conformational changes that prevent activation of SREBPs and halting synthesis of lipids. To achieve a molecular understanding of how cholesterol regulates the Scap/SREBP machine and to identify therapeutics for dysregulated lipid metabolism, cholesterol-mimetic compounds that specifically bind and inhibit Scap are needed. To accomplish this goal, we focused on Anthrolysin O (ALO), a pore-forming bacterial toxin that binds cholesterol with a specificity and sensitivity that is uncannily similar to Scap. We reasoned that a small molecule that would bind and inhibit ALO might also inhibit Scap. High-throughput screening of a ~300,000-compound library for ALO-binding unearthed one molecule, termed UT-59, which binds to Scap's cholesterol-binding site. Upon binding, UT-59 triggers the same conformation changes in Scap as those induced by cholesterol and blocks activation of SREBPs and lipogenesis in cultured cells. UT-59 also inhibits SREBP activation in the mouse liver. Unlike five previously reported inhibitors of SREBP activation, UT-59 is the only one that acts specifically by binding to Scap's cholesterol-binding site. Our approach to identify specific Scap inhibitors such as UT-59 holds great promise in developing therapeutic leads for human diseases stemming from elevated SREBP activation, such as fatty liver and certain cancers.

Palmitoylation of the Glucagon-like Peptide-1 Receptor Modulates Cholesterol Interactions at the Receptor-Lipid Microenvironment.

The G protein-coupled receptor (GPCR) superfamily of cell surface receptors has been shown to be functionally modulated by post-translational modifications. The glucagon-like peptide receptor-1 (GLP-1R), which is a drug target in diabetes and obesity, undergoes agonist-dependent palmitoyl tail conjugation. The palmitoylation in the C-terminal domain of GLP-1R has been suggested to modulate the receptor-lipid microenvironment. In this work, we have performed coarse-grain molecular dynamics simulations of palmitoylated and nonpalmitoylated GLP-1R to analyze the differential receptor-lipid interactions. Interestingly, the placement and dynamics of the C-terminal domain of GLP-1R are found to be directly dependent on the palmitoyl tail. We observe that both cholesterol and phospholipids interact with the receptor but display differential interactions in the presence and absence of the palmitoyl tail. We characterize important cholesterol-binding sites and validate sites that have been previously reported in experimentally resolved structures of the receptor. We show that the receptor acts like a conduit for cholesterol flip-flop by stabilizing cholesterol in the membrane core. Taken together, our work represents an important step in understanding the molecular effects of lipid modifications in GPCRs.

Biophysical Analysis to Assess the Interaction of CRAC and CARC Motif Peptides of Alpha Hemolysin of Escherichia coli with Membranes.

Alpha hemolysin of Escherichia coli (HlyA) is a pore-forming protein, which is a prototype of the "Repeat in Toxins" (RTX) family. It was demonstrated that HlyA-cholesterol interaction facilitates the insertion of the toxin into membranes. Putative cholesterol-binding sites, called cholesterol recognition/amino acid consensus (CRAC), and CARC (analogous to CRAC but with the opposite orientation) were identified in the HlyA sequence. In this context, two peptides were synthesized, one derived from a CARC site from the insertion domain of the toxin (residues 341-353) (PEP 1) and the other one from a CRAC site from the domain between the acylated lysines (residues 639-644) (PEP 2), to study their role in the interaction of HlyA with membranes. The interaction of peptides with membranes of different lipid compositions (pure POPC and POPC/Cho of 4:1 and 2:1 molar ratios) was analyzed by surface plasmon resonance and molecular dynamics simulations. Results demonstrate that both peptides interact preferentially with Cho-containing membranes, although PEP 2 presents a lower KD than PEP 1. Molecular dynamics simulation results indicate that the insertion and interaction of PEP 2 with Cho-containing membranes are more prominent than those caused by PEP 1. The hemolytic activity of HlyA in the presence of peptides indicates that PEP 2 was the only one that inhibits HlyA activity, interfering in the binding between the toxin and cholesterol.

Allosterically coupled conformational dynamics in solution prepare the sterol transfer protein StarD4 to release its cargo upon interaction with target membranes.

Complex mechanisms regulate the cellular distribution of cholesterol, a critical component of eukaryote membranes involved in regulation of membrane protein functions directly and through the physiochemical properties of membranes. StarD4, a member of the steroidogenic acute regulator-related lipid-transfer (StART) domain (StARD)-containing protein family, is a highly efficient sterol-specific transfer protein involved in cholesterol homeostasis. Its mechanism of cargo loading and release remains unknown despite recent insights into the key role of phosphatidylinositol phosphates in modulating its interactions with target membranes. We have used large-scale atomistic Molecular dynamics (MD) simulations to study how the dynamics of cholesterol bound to the StarD4 protein can affect interaction with target membranes, and cargo delivery. We identify the two major cholesterol (CHL) binding modes in the hydrophobic pocket of StarD4, one near S136&S147 (the Ser-mode), and another closer to the putative release gate located near W171, R92&Y117 (the Trp-mode). We show that conformational changes of StarD4 associated directly with the transition between these binding modes facilitate the opening of the gate. To understand the dynamics of this connection we apply a machine-learning algorithm for the detection of rare events in MD trajectories (RED), which reveals the structural motifs involved in the opening of a front gate and a back corridor in the StarD4 structure occurring together with the spontaneous transition of CHL from the Ser-mode of binding to the Trp-mode. Further analysis of MD trajectory data with the information-theory based NbIT method reveals the allosteric network connecting the CHL binding site to the functionally important structural components of the gate and corridor. Mutations of residues in the allosteric network are shown to affect the performance of the allosteric connection. These findings outline an allosteric mechanism which prepares the CHL-bound StarD4 to release and deliver the cargo when it is bound to the target membrane.

New molecular insights into the A218V variant impact on the steroidogenic acute regulatory protein (STAR) associated with 46, XY disorders of sexual development

Disorders of sexual development (DSD) are an abnormal congenital conditions associated with atypical development of the urogenital tract and external genital structures. The steroidogenic acute regulatory (STAR) gene, associated with congenital lipoid adrenal hyperplasia (CLAH), is included in the targeted gene panel for the DSD diagnosis. Therefore, the genetic alterations of the STAR gene and their molecular effect were examined in the CLAH patients affected with DSD. Ten different Iranian families including twelve male pseudo-hermaphroditism patients with CLAH phenotype were studied using genetic linkage screening and STAR gene sequencing in the linked families to the STAR locus. Furthermore, the structural, dynamical, and functional impacts of the variants on the STAR in silico were analyzed. Sanger sequencing showed the pathogenic variant p.A218V in STAR gene, as the first report in Iranian population. Moreover, modeling and simulation analysis were performed using tools such as radius of gyration, root mean square deviation (RMSD), root mean square fluctuation (RMSF), and molecular docking showed that p.A218V variant affects the residues interaction in cholesterol-binding site and the proper folding of STAR through increasing H-bound and the amount of α-Helix, deceasing total flexibility and changing fluctuations in some residues, resulting in reduced steroidogenic activity of the STAR protein. The study characterized the structural and functional changes of STAR caused by pathogenic variant p.A218V. It leads to limited cholesterol-binding activity of STAR, ultimately leading to the CLAH disease. Molecular dynamics simulation of STAR variants could help explain different clinical manifestations of CLAH disease.

Docking cholesterol to integral membrane proteins with Rosetta.

Lipid molecules such as cholesterol interact with the surface of integral membrane proteins (IMP) in a mode different from drug-like molecules in a protein binding pocket. These differences are due to the lipid molecule's shape, the membrane's hydrophobic environment, and the lipid's orientation in the membrane. We can use the recent increase in experimental structures in complex with cholesterol to understand protein-cholesterol interactions. We developed the RosettaCholesterol protocol consisting of (1) a prediction phase using an energy grid to sample and score native-like binding poses and (2) a specificity filter to calculate the likelihood that a cholesterol interaction site may be specific. We used a multi-pronged benchmark (self-dock, flip-dock, cross-dock, and global-dock) of protein-cholesterol complexes to validate our method. RosettaCholesterol improved sampling and scoring of native poses over the standard RosettaLigand baseline method in 91% of cases and performs better regardless of benchmark complexity. On the β2AR, our method found one likely-specific site, which is described in the literature. The RosettaCholesterol protocol quantifies cholesterol binding site specificity. Our approach provides a starting point for high-throughput modeling and prediction of cholesterol binding sites for further experimental validation.

Comparative Study of Receptor-, Receptor State-, and Membrane-Dependent Cholesterol Binding Sites in A2A and A1 Adenosine Receptors Using Coarse-Grained Molecular Dynamics Simulations.

We used coarse-grained molecular dynamics (CG MD) simulations to study protein-cholesterol interactions for different activation states of the A2A adenosine receptor (A2AR) and the A1 adenosine receptor (A1R) and predict new cholesterol binding sites indicating amino acid residues with a high residence time in three biologically relevant membranes. Compared to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)-cholesterol and POPC-phosphatidylinositol-bisphosphate (PIP2)-cholesterol, the plasma mimetic membrane best described the cholesterol binding sites previously detected for the inactive state of A2AR and revealed the binding sites with long-lasting amino acid residues. We observed that using the plasma mimetic membrane and plotting residues with cholesterol residence time ≥2 μs, our CG MD simulations captured most obviously the cholesterol-protein interactions. For the inactive A2AR, we identified one more binding site in which cholesterol is bound to residues with a long residence time compared to the previously detected, for the active A1R, three binding sites, and for the inactive A1R, two binding sites. We calculated that for the active states, cholesterol binds to residues with a much longer residence time compared to the inactive state for both A2AR and A1R. The stability of the identified binding sites to A1R or A2AR with CG MD simulations was additionally investigated with potential of mean force calculations using umbrella sampling. We observed that the binding sites with residues to which cholesterol has a long residence time in A2AR have shallow binding free energy minima compared to the related binding sites in A1R, suggesting a stronger binding for cholesterol to A1R. The differences in binding sites in which cholesterol is stabilized and interacts with residues with a long residence time between active and inactive states of A1R and A2AR can be important for differences in functional activity and orthosteric agonist or antagonist affinity and can be used for the design of allosteric modulators, which can bind through lipid pathways. We observed a stronger binding for cholesterol to A1R (i.e., generally higher association rates) compared to A2AR, which remains to be demonstrated. For the active states, cholesterol binds to residues with much longer residence times compared to the inactive state for both A2AR and A1R. Taken together, binding sites of active A1R may be considered as promising allosteric targets.

Specific Mutations in the Cholesterol-Binding Site of APP Alter Its Processing and Favor the Production of Shorter, Less Toxic Aβ Peptides

Excess brain cholesterol is strongly implicated in the pathogenesis of Alzheimer’s disease (AD). Here we evaluated how the presence of a cholesterol-binding site (CBS) in the transmembrane and juxtamembrane regions of the amyloid precursor protein (APP) regulates its processing. We generated nine point mutations in the APP gene, changing the charge and/or hydrophobicity of the amino-acids which were previously shown as part of the CBS. Most mutations triggered a reduction of amyloid-β peptides Aβ40 and Aβ42 secretion from transiently transfected HEK293T cells. Only the mutations at position 28 of Aβ in the APP sequence resulted in a concomitant significant increase in the production of shorter Aβ peptides. Mass spectrometry (MS) confirmed the predominance of Aβx-33 and Aβx-34 with the APPK28A mutant. The enzymatic activity of α-, β-, and γ-secretases remained unchanged in cells expressing all mutants. Similarly, subcellular localization of the mutants in early endosomes did not differ from the APPWT protein. A transient increase of plasma membrane cholesterol enhanced the production of Aβ40 and Aβ42 by APPWT, an effect absent in APPK28A mutant. Finally, WT but not CBS mutant Aβ derived peptides bound to cholesterol-rich exosomes. Collectively, the present data revealed a major role of juxtamembrane amino acids of the APP CBS in modulating the production of toxic Aβ species. More generally, they underpin the role of cholesterol in the pathophysiology of AD.

Exploring TRPC3 Interaction with Cholesterol through Coarse-Grained Molecular Dynamics Simulations.

Transient receptor potential canonical 3 (TRPC3) channel belongs to the superfamily of transient receptor potential (TRP) channels which mediate Ca2+ influx into the cell. These channels constitute essential elements of cellular signalling and have been implicated in a wide range of diseases. TRPC3 is primarily gated by lipids and its surface expression has been shown to be dependent on cholesterol, yet a comprehensive exploration of its interaction with this lipid has thus far not emerged. Here, through 80 µs of coarse-grained molecular dynamics simulations, we show that cholesterol interacts with multiple elements of the transmembrane machinery of TRPC3. Through our approach, we identify an annular binding site for cholesterol on the pre-S1 helix and a non-annular site at the interface between the voltage-sensor-like domain and pore domains. Here, cholesterol interacts with exposed polar residues and possibly acts to stabilise the domain interface.

Hepatocyte cholesterol content modulates glucagon receptor signalling

Structured abstractObjectiveTo determine whether glucagon receptor (GCGR) actions are modulated by cellular cholesterol levels.MethodsWe determined the effects of experimental cholesterol depletion and loading on glucagon-mediated cAMP production, ligand internalisation and glucose production in human hepatoma cells, mouse and human hepatocytes. GCGR interactions with lipid bilayers were explored using coarse-grained molecular dynamic simulations. Glucagon responsiveness was measured in mice fed a high cholesterol diet with or without simvastatin to modulate hepatocyte cholesterol content.ResultsGCGR cAMP signalling was reduced by higher cholesterol levels across different cellular models. Ex vivo glucagon-induced glucose output from mouse hepatocytes was enhanced by simvastatin treatment. Mice fed a high cholesterol diet had increased hepatic cholesterol and a blunted hyperglycaemic response to glucagon, both of which were partially reversed by simvastatin. Simulations identified likely membrane-exposed cholesterol binding sites on the GCGR, including a site where cholesterol is a putative negative allosteric modulator.ConclusionsOur results indicate that cellular cholesterol content influences glucagon sensitivity and indicate a potential molecular basis for this phenomenon. This could be relevant to the pathogenesis of non-alcoholic fatty liver disease, which is associated with both hepatic cholesterol accumulation and glucagon resistance.

Role of ACAT1 in Atherosclerosis and Alzheimer's Disease

The acyl‐coenzyme A: cholesterol acyltransferase (ACAT) intracellular membrane protein is located in the endoplasmic reticulum and is composed of 427 amino acids. ACAT is a part of the membrane‐bound O‐acyltransferase enzyme family (MBOAT). MBOATs are recognized by their most carried residues, histamine, and arginine, and are used in lipid synthesis, phospholipid remodeling, and peptide acylation. The enzyme itself is structured as a tetramer split into two homodimers. Nine transmembrane helices or domains (TMs) are on each monomer, the most notable being a cavity formed through TMs 4‐9 harboring histidine, an amino acid tied to catalysis and enzymatic activity.The ACAT1 protein‐coupled with its ACAT2 counterpart catalyze the storage of cholesteryl esters. ACAT2 differs from ACAT1 in function and structure. ACAT2 contains 2 domains and is mainly expressed in intestinal cells and controls lipoprotein particle secretion. The enzyme transfers an acyl group to 3β‐hydroxyl of cholesterol, a cholesterol‐binding site, to form cytosolic lipid droplets that are used for reducing membrane fluidity of the cell. Lipo‐protein‐induced ACAT in microphages is key to the creation of atheroma foam cells, cells characterized by the aggregation of these fatty plaque deposits on the walls of arteries, thus appearing like foam. The aggregation of this plaque to eventually narrow and harden arteries to obstruct blood flow is atherosclerosis. ACAT1 also maintains homeostasis during periods of cholesterol overload in the ER. Other functions of the protein include aiding lipoprotein synthesis and dietary LDL absorption.Recent studies have seen a pathogenic connection between cholesterol and Alzheimer’s disease or other neurodegenerative diseases. Cells contain a membrane that contains microdomains rich in cholesterol. The microdomains assist in the maintenance and growth of the cell and cell membrane; however, it is the disruption of these microdomains through individual disease‐causing toxins that cause them to inactivate ACAT1 and block cholesterol storage, disrupting brain activity.ACAT is currently being studied for its targeted drug therapy countering atherosclerosis. Current studies have proved ACAT inhibition to be ineffective, but there still remains potential immunotherapies to neurodegenerative disorders through the ACAT enzyme. Further research remains to be done to analyze the link between neurodegenerative disorders and the ACAT1 protein.

Discovery of the first tight‐binding reversible antagonists of Hedgehog protein autoprocessing

Hedgehog (Hh) signaling ligands (HhN) are expressed as precursors with an adjacent C‐terminal autoprocessing domain, HhC. Autoprocessing activity of HhC releases and cholesterylates the Hh ligand thereby activating the HhN for full biological activity. Inhibitors of HhC activity are of interest as probes of the Hh signaling pathway, and HhC inhibitors may also offer new therapeutic approaches to suppress oncogenic signaling activity of HhN. The intramolecular nature of the HhN/HhC system poses a challenge for reversible inhibitors, and to date most antagonists of HhC bind the active site covalently. Here we report we report the discovery and optimization of the first tight‐binding reversible inhibitor of HhC. Using a light‐to‐dark FRET activity assay for Drosophila melanogaster HhC, we screened a focused library of 1187 sterol‐like analogs in search of HhC inhibitors. Four structurally related hits were identified and further validated by dose‐response assays. The most effective inhibitor from the screen had an IC50 value of 2x10‐6M. A panel of analogs were prepared and tested for SAR analysis resulting in compound tBT‐HBT, a derivative with a sub‐micromolar IC50of 3x10‐7 M. Inhibition appears to be mediated by a previously unidentified allosteric binding site on HhC. Kinetic analysis, photoaffinity labeling, computational modeling, and point mutagenesis studies suggest that the binding site is nearby, but not overlapping with the cholesterol binding site of HhC.