Regulation of the Ca2+-ATPase by cholesterol: A specific or non-specific effect?

Abstract

Like other integral membrane proteins, the activity of the Sarco/Endoplasmic Reticulum Ca²⁺-ATPase (SERCA) is regulated by the membrane environment. Cholesterol is present in the endoplasmic reticulum membrane at low levels, and it has the potential to affect SERCA activity both through direct, specific interaction with the protein or through indirect interaction through changes of the overall membrane properties. There are experimental data arguing for both modes of action for a cholesterol-mediated regulation of SERCA. In the current study, coarse-grained molecular dynamics simulations are used to address how a mixed lipid-cholesterol membrane interacts with SERCA. Candidates for direct regulatory sites with specific cholesterol binding modes are extracted from the simulations. The binding pocket for thapsigargin, a nanomolar inhibitor of SERCA, has been suggested as a cholesterol binding site. However, the thapsigargin binding pocket displayed very little cholesterol occupation in the simulations. Neither did atomistic simulations of cholesterol in the thapsigargin binding pocket support any specific interaction. The current study points to a non-specific effect of cholesterol on SERCA activity, and offers an alternative interpretation of the experimental results used to argue for a specific effect.     

The function of G-protein coupled receptors and membrane cholesterol: specific or general interaction?

Cholesterol is an essential component of eukaryotic membranes and plays a crucial role in membrane organization, dynamics and function. The G-protein coupled receptors (GPCRs) are the largest class of molecules involved in signal transduction across membranes and constitute ~1–2% of the human genome. GPCRs have emerged as major targets for the development of novel drug candidates in all clinical areas due to their involvement in the generation of multitude of cellular responses. Membrane cholesterol has been reported to have a modulatory role in the function of a number of GPCRs. This effect could either be due to specific molecular interaction between cholesterol and GPCR, or due to alterations in the membrane physical properties induced by cholesterol. Alternatively, membrane cholesterol could modulate receptor function by occupying the ‘nonannular’ sites around the receptor. In this review, we have highlighted the nature of cholesterol dependence of GPCR function taking a few known examples.     

Pharmacological inhibition of the intracellular transport of low-density lipoprotein-derived cholesterol in Chinese hamster ovary cells

Mammalian cells, cultured in the presence of serum lipoproteins, acquire cholesterol necessary for growth from the uptake and lysosomal hydrolysis of low-density lipoproteins (LDL). The mechanism(s) of intracellular transport of LDL-derived cholesterol from lysosomes to other cellular sites is unknown. In this study, various pharmacological agents were assessed for their ability to inhibit the movement of LDL-cholesterol from lysosomes to the plasma membrane. The only pharmacological agent tested in these experiments that specifically inhibited LDL-cholesterol movement was U18666A. Ketoconazole impaired the intracellular transport of LDL-cholesterol; however, ketoconazole also had a general effect on cholesterol movement, since it impeded the desorption of endogenously synthesized cholesterol into the medium. Other drugs that affected cholesterol movement appeared to be nonspecific. Cholesterol transport from lysosomes to plasma membranes was not significantly altered by agents that affect lysosomal function or cytoskeletal organization, as well as energy poisons and cycloheximide.     

Molecular modelling study of the role of cholesterol in the stimulation of the oxytocin receptor.

Cholesterol, an integral component of membranes in Eucaryota, is a modifier of membrane properties. In vivo studies have demonstrated that cholesterol can also modulate activities of some G protein-coupled receptors (GPCRs), which are integral membrane proteins. This can result either from an effect of cholesterol on the membrane fluidity or from specific interactions of the membrane cholesterol with the receptor, as recently demonstrated for the cholecystokinin type beta (CCKRbeta) or the oxytocin receptor (OTR). Using molecular modelling, we studied conformational preferences of cholesterol and several of its analogues. Subsequently, we simulated the distributions of their preferred conformations around the surface of OTR, CCKRbeta and a chimeric oxytocin/cholecystokinin receptor. Consequently, we suggest residues on the surface of OTR which are potentially significant in the OTR/cholesterol interaction.     

Tuning of the outer hair cell motor by membrane cholesterol

Cholesterol affects diverse biological processes, in many cases by modulating the function of integral membrane proteins. We observed that alterations of cochlear cholesterol modulate hearing in mice. Mammalian hearing is powered by outer hair cell (OHC) electromotility, a membrane-based motor mechanism that resides in the OHC lateral wall. We show that membrane cholesterol decreases during maturation of OHCs. To study the effects of cholesterol on hearing at the molecular level, we altered cholesterol levels in the OHC wall, which contains the membrane protein prestin. We show a dynamic and reversible relationship between membrane cholesterol levels and voltage dependence of prestin-associated charge movement in both OHCs and prestin-transfected HEK 293 cells. Cholesterol levels also modulate the distribution of prestin within plasma membrane microdomains and affect prestin self-association in HEK 293 cells. These findings indicate that alterations in membrane cholesterol affect prestin function and functionally tune the outer hair cell.     

A simple and rapid method to measure cholesterol binding to P450s and other proteins

Cholesterol plays an important role in cellular function and membrane compartmentalization and is involved in the interaction with more than a dozen of different proteins. Using three cholesterol-metabolizing cytochrome P450s (P450s 7A1, 46A1, and 11A1), we have developed a rapid and simple assay for measurements of nanomolar to micromolar cholesterol affinities. In this assay, the P450 is incubated with a fixed amount of radiolabeled cholesterol and varying concentrations of cold cholesterol followed by separation of free and protein-bound cholesterol via filtration through a membrane. Free cholesterol is found in the flow-through fraction, whereas P450 binds to the membrane. The radioactivity of the membranes is then measured, and a saturation curve is generated after correction for nonspecific binding of cholesterol to the filter. The validity of the filter assay was confirmed by spectral assay, a traditional method to evaluate the interaction of the P450 enzymes with their substrates. Two types of membranes, one binding positively charged proteins and another binding negatively charged proteins, were identified. These membranes were also found to hold proteins through hydrophobic interactions. Thus, the cholesterol binding properties of a wide variety of proteins could be characterized using this filter assay.     

Putative cholesterol‐binding sites in human immunodeficiency virus (HIV) coreceptors CXCR4 and CCR5

Using molecular docking, we identified a cholesterol‐binding site in the groove between transmembrane helices 1 and 7 near the inner membrane‐water interface of the G protein‐coupled receptor CXCR4, a coreceptor for HIV entry into cells. In this docking pose, the amino group of lysine K67 establishes a hydrogen bond with the hydroxyl group of cholesterol, whereas tyrosine Y302 stacks with cholesterol by its aromatic side chain, and a number of residues form hydrophobic contacts with cholesterol. Sequence alignment showed that a similar putative cholesterol‐binding site is also present in CCR5, another HIV coreceptor. We suggest that the interaction of cholesterol with these putative cholesterol‐binding sites in CXCR4 and CCR5 is responsible for the presence of these receptors in lipid rafts, for the effect of cholesterol on their conformational stability and function, and for the role that cell cholesterol plays in the cell entry of HIV strains that use these membrane proteins as coreceptors. We propose that mutations of residues that are involved in cholesterol binding will make CXCR4 and CCR5 insensitive to membrane cholesterol content. Cholesterol‐binding sites in HIV coreceptors are potential targets for steroid drugs that bind to CXCR4 and CCR5 with higher binding affinity than cholesterol, but do not stabilize the native conformation of these proteins. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Role of cholesterol in the function and organization of G-protein coupled receptors

Cholesterol is an essential component of eukaryotic membranes and plays a crucial role in membrane organization, dynamics and function. The modulatory role of cholesterol in the function of a number of membrane proteins is well established. This effect has been proposed to occur either due to a specific molecular interaction between cholesterol and membrane proteins or due to alterations in the membrane physical properties induced by the presence of cholesterol. The contemporary view regarding heterogeneity in cholesterol distribution in membrane domains that sequester certain types of membrane proteins while excluding others has further contributed to its significance in membrane protein function. The seven transmembrane domain G-protein coupled receptors (GPCRs) are among the largest protein families in mammals and represent approximately 2% of the total proteins coded by the human genome. Signal transduction events mediated by this class of proteins are the primary means by which cells communicate with and respond to their external environment. GPCRs therefore represent major targets for the development of novel drug candidates in all clinical areas. In view of their importance in cellular signaling, the interaction of cholesterol with such receptors represents an important determinant in functional studies of such receptors. This review focuses on the effect of cholesterol on the membrane organization and function of GPCRs from a variety of sources, with an emphasis on the more contemporary role of cholesterol in maintaining a domain-like organization of such receptors on the cell surface. Importantly, the recently reported role of cholesterol in the function and organization of the neuronal serotonin(1A) receptor, a representative of the GPCR family which is present endogenously in the hippocampal region of the brain, will be highlighted.     

The Physical and Genetic Organization of A Viral Genom

Membrane proteins that bind and transport lipids face special challenges. Since lipids typically have low water solubility, both accessibility of the substrate to the protein and delivery to the desired destination are problematical. The amphipathic nature of membrane lipids, and their relatively large molecular size, also means that these proteins must possess substrate-binding sites of a different nature than those designed to handle small polar molecules. This review considers two integral proteins whose function is to bind and transfer membrane lipids within or across a membrane. The first protein, MsbA, is a putative lipid flippase that is a member of the ATP-binding cassette (ABC) superfamily. The protein is found in the inner (cytoplasmic) membrane (IM) of Gram-negative bacteria such as E. coli, where it is proposed to move lipid A from the inner to the outer membrane (OM) leaflet, an important step in the lipopolysaccharide biosynthetic pathway. Cholesterol is a major component of the plasma membrane in eukaryotic cells, where it regulates bilayer fluidity. The other lipid-binding protein discussed here, mammalian NPC1 (Niemann-Pick disease, Type C1), binds cholesterol inside late endosomes/lysosomes (LE/LY) and is involved in its transfer to the cytosol as part of a key intracellular sterol-trafficking pathway. Mutations in NPC1 lead to a devastating neurodegenerative condition, Niemann-Pick Type C disease, which is characterized by massive cholesterol accumulation in LE/LY. The accelerating pace of membrane protein structure determination over the past decade has allowed us a glimpse of how lipid binding and transfer by membrane proteins such as MsbA and NPC1 might be achieved.     

Niemann–Pick C2 (NPC2) and intracellular cholesterol trafficking

Cholesterol is an important precursor for numerous biologically active molecules, and it plays a major role in membrane structure and function. Cholesterol can be endogenously synthesized or exogenously taken up via the endocytic vesicle system and subsequently delivered to post-endo/lysosomal sites including the plasma membrane and the endoplasmic reticulum. Niemann–Pick C (NPC) disease results in the accumulation of exogenously-derived cholesterol, as well as other lipids, in late endosomes and lysosomes (LE/LY). Identification of the two genes that underlie NPC disease, NPC1 and NPC2, has focused attention on the mechanisms by which lipids, in particular cholesterol, are transported out of the LE/LY compartment. This review discusses the role of the NPC2 protein in cholesterol transport, and the potential for concerted action of NPC1 and NPC2 in regulating normal intracellular cholesterol homeostasis.     

Modulation of membrane function by cholesterol.

The molecular basis for the essential role of cholesterol in mammalian (and other cholesterol-requiring) cells has long been the object of intense interest. Cholesterol has been found to modulate the function of membrane proteins critical to cellular function. Current literature supports two mechanisms for this modulation. In one mechanism, the requirement of 'free volume' by integral membrane proteins for conformational changes as part of their functional cycle is antagonized by the presence of high levels of cholesterol in the membrane. In the other mechanism, the sterol modulates membrane protein function through direct sterol-protein interactions. This mechanism provides an explanation for the stimulation of the activity of important membrane proteins and for the essential requirement of a structurally-specific sterol for cell viability. In some cases, these latter membrane proteins exhibit little or no activity in the absence of the specific sterol required for growth of that cell type. The specific sterol required varies from one cell type to another and is unrelated to the ability of that sterol to affect the bulk properties of the membrane.     

Spontaneous cholesterol movement between lipid vesicles and monkey small intestinal brush border membrane

[14C]Cholesterol movement between egg phosphatidylcholine-cholesterol lipid vesicles and vesicles prepared from monkey small intestinal brush border membrane (BBMV) was studied in physiological buffer at 37 degrees C. The rate of cholesterol transfer from sonicated unilamellar vesicles (ULV) to BBMV follows apparently first-order kinetics. Intermembrane cholesterol movement was strikingly similar in both the directions. However, from BBMV to ULV, the transfer rate was three times faster than that of ULV to brush border membrane (BBM). Similarity in the rate constant was observed when cholesterol transfer was studied using either large multilamellar lipid vesicles or ULV as the donor and BBMV as the acceptor membrane. Rate constant was also the same when the acceptor membrane used was either intact BBMV or ULV prepared from BBM lipids. The rate of transfer of label was not affected even when the acceptor vesicle concentration was increased over fivefold, indicating the first-order nature of the reaction. Transfer of cholesterol from ULV to BBMV was accelerated by the presence of acetone, dimethyl sulfoxide (DMSO), deoxycholate, and papain. Partially purified nonspecific lipid-exchange protein increased the rate of cholesterol transfer by about threefold. Reduction in BBM cholesterol and phospholipid content was noted by DMSO, acetone, and deoxycholate, while papain caused a small depletion of membrane protein. Cholesterol transfer is temperature dependent with an activation energy of 31 kJ X mol-1, which is almost identical in the presence or absence of nonspecific lipid-exchange protein. The molecular mechanism of intermembrane cholesterol movement is discussed in view of the kinetic data obtained.     

In vitro studies on the role of the peripheral-type benzodiazepine receptor in steroidogenesis

In vitro studies using isolated cells, mitochondria and submitochondrial fractions demonstrated that in steroid synthesizing cells, the peripheral-type benzodiazepine receptor (PBR) is an outer mitochondrial membrane protein, preferentially located in the outer/inner membrane contact sites, involved in the regulation of cholesterol transport from the outer to the inner mitochondrial membrane, the rate-determining step in steroid biosynthesis. Mitochondrial PBR ligand binding characteristics and topography are sensitive to hormone treatment suggesting a role of PBR in the regulation of hormone-mediated steroidogenesis. Targeted disruption of the PBR gene in Leydig cells in vitro resulted in the arrest of cholesterol transport into mitochondria and steroid formation; transfection of the mutant cells with a PBR cDNA rescued steroidogenesis demonstrating an obligatory role for PBR in cholesterol transport. Molecular modeling of PBR suggested that it might function as a channel for cholesterol. This hypothesis was tested in a bacterial system devoid of PBR and cholesterol. Cholesterol uptake and transport by these cells was induced upon PBR expression. Amino acid deletion followed by site-directed mutagenesis studies and expression of mutant PBRs demonstrated the presence in the cytoplasmic carboxy-terminus of the receptor of a cholesterol recognition/interaction amino acid consensus sequence. This amino acid sequence may help for recruiting the cholesterol coming from intracellular sites to the mitochondria.     

Proteins that bind and move lipids: MsbA and NPC1

Membrane proteins that bind and transport lipids face special challenges. Since lipids typically have low water solubility, both accessibility of the substrate to the protein and delivery to the desired destination are problematical. The amphipathic nature of membrane lipids, and their relatively large molecular size, also means that these proteins must possess substrate-binding sites of a different nature than those designed to handle small polar molecules. This review considers two integral proteins whose function is to bind and transfer membrane lipids within or across a membrane. The first protein, MsbA, is a putative lipid flippase that is a member of the ATP-binding cassette (ABC) superfamily. The protein is found in the inner (cytoplasmic) membrane (IM) of Gram-negative bacteria such as E. coli, where it is proposed to move lipid A from the inner to the outer membrane (OM) leaflet, an important step in the lipopolysaccharide biosynthetic pathway. Cholesterol is a major component of the plasma membrane in eukaryotic cells, where it regulates bilayer fluidity. The other lipid-binding protein discussed here, mammalian NPC1 (Niemann-Pick disease, Type C1), binds cholesterol inside late endosomes/lysosomes (LE/LY) and is involved in its transfer to the cytosol as part of a key intracellular sterol-trafficking pathway. Mutations in NPC1 lead to a devastating neurodegenerative condition, Niemann-Pick Type C disease, which is characterized by massive cholesterol accumulation in LE/LY. The accelerating pace of membrane protein structure determination over the past decade has allowed us a glimpse of how lipid binding and transfer by membrane proteins such as MsbA and NPC1 might be achieved.     

Are specific nonannular cholesterol binding sites present in G-protein coupled receptors?

The G-protein coupled receptors (GPCRs) are the largest class of molecules involved in signal transduction across membranes, and represent major drug targets in all clinical areas. Membrane cholesterol has been reported to have a modulatory role in the function of a number of GPCRs. Interestingly, recently reported crystal structures of GPCRs have shown structural evidence of cholesterol binding sites. Two possible mechanisms have been previously suggested by which membrane cholesterol could influence the structure and function of GPCRs (i) through a direct/specific interaction with GPCRs, which could induce a conformational change in the receptor, or (ii) through an indirect way by altering the membrane physical properties in which the receptor is embedded or due to a combination of both. We discuss here a novel mechanism by which membrane cholesterol could affect structure and function of GPCRs and propose that cholesterol binding sites in GPCRs could represent ‘nonannular’ binding sites. Interestingly, previous work from our laboratory has demonstrated that membrane cholesterol is required for the function of the serotonin_1A receptor, which could be due to specific interaction of the receptor with cholesterol. Based on these results, we envisage that there could be specific/nonannular cholesterol binding site(s) in the serotonin_1A receptor. We have analyzed putative cholesterol binding sites from protein databases in the serotonin_1A receptor, a representative GPCR, for which we have previously demonstrated specific requirement of membrane cholesterol for receptor function. Our analysis shows that cholesterol binding sites are inherent characteristic features of serotonin_1A receptors and are conserved over evolution. Progress in deciphering molecular details of the nature of GPCR-cholesterol interaction in the membrane would lead to better insight into our overall understanding of GPCR function in health and disease, thereby enhancing our ability to design better therapeutic strategies to combat diseases related to malfunctioning of GPCRs.     

Staying in touch with the endocytic network: The importance of contacts for cholesterol transport

Cholesterol homeostasis is critical for cell function and human health. Cholesterol is heterogeneously distributed among cellular membranes, with the redistribution of endocytosed dietary cholesterol playing a pivotal role in the regulation of cholesterol homeostasis. While gaps remain in our understanding of intracellular dietary cholesterol transport, a highly complex network of pathways is starting to emerge, often involving inter‐dependent vesicular and non‐vesicular transport mechanisms. The last decade has seen a surge in interest in non‐vesicular transport and inter‐organellar communication at membrane contact sites. By providing platforms for protein interactions, signalling events, lipid exchange and calcium flux, membrane contact sites (MCS) are now appreciated as controlling the fate of large amounts of lipid and play central roles in the regulation and co‐ordination of endocytic trafficking. Here, we review the role of MCS in multiple pathways for cholesterol export from the endocytic pathway and highlight the intriguing interplay between vesicular and non‐vesicular transport mechanisms and relationship with neurodegenerative disease.     

Two Classes of Cholesterol Binding Sites for the β2AR Revealed by?Thermostability and NMR

Cholesterol binding to G protein-coupled receptors (GPCRs) and modulation of their activities in membranes is a fundamental issue for understanding their function. Despite the identification of cholesterol binding sites in high-resolution x-ray structures of the β₂ adrenergic receptor (β₂AR) and other GPCRs, the binding affinity of cholesterol for this receptor and exchange rates between the free and bound cholesterol remain unknown. In this study we report the existence of two classes of cholesterol binding sites in β₂AR. By analyzing the β₂AR unfolding temperature in lipidic cubic phase (LCP) as a function of cholesterol concentration we observed high-affinity cooperative binding of cholesterol with sub-nM affinity constant. In contrast, saturation transfer difference (STD) NMR experiments revealed the existence of a second class of cholesterol binding sites, in fast exchange on the STD NMR timescale. Titration of the STD signal as a function of cholesterol concentration provided a lower limit of 100 mM for their dissociation constant. However, these binding sites are specific for both cholesterol and β₂AR, as shown with control experiments using ergosterol and a control membrane protein (KpOmpA). We postulate that this specificity is mediated by the high-affinity bound cholesterol molecules and propose the formation of transient cholesterol clusters around the high-affinity binding sites.     

Cholesterol modulates alkaline phosphatase activity of rat intestinal microvillus membranes

Experiments were conducted, using a nonspecific lipid transfer protein, to vary the cholesterol/phospholipid molar ratio of rat proximal small intestinal microvillus membranes in order to assess the possible role of cholesterol in modulating enzymatic activities of this plasma membrane. Cholesterol/phospholipid molar ratios from 0.71 to 1.30 were produced from a normal value of 1.05 by incubation with the transfer protein and an excess of either phosphatidylcholine or cholesterol/phosphatidylcholine liposomes for 60 min at 37 degrees C. Cholesterol loading or depletion of the membranes was accompanied by a decrease or increase, respectively, in their lipid fluidity, as assessed by steady-state fluorescence polarization techniques using the lipid-soluble fluorophore 1,6-diphenyl-1,3,5-hexatriene. Increasing the cholesterol/phospholipid molar ratio also decreased alkaline phosphatase specific activity by approximately 20-30%, whereas decreasing this ratio increased this enzymatic activity by 20-30%. Sucrase, maltase, and lactase specific activities were not affected in these same preparations. Since the changes in alkaline phosphatase activity could be secondary to alterations in fluidity, cholesterol, or both, additional experiments were performed using benzyl alcohol, a known fluidizer. Benzyl alcohol (25 mM) restored the fluidity of cholesterol-enriched preparations to control levels, did not change the cholesterol/phospholipid molar ratio, and failed to alter alkaline phosphatase activity. These findings, therefore, indicate that alterations in the cholesterol content and cholesterol/phospholipid molar ratio of microvillus membranes can modulate alkaline phosphatase but not sucrase, maltase, or lactase activities. Moreover, membrane fluidity does not appear to be an important physiological regulator of these enzymatic activities.     

Duck hepatitis B virus requires cholesterol for endosomal escape during virus entry

The identity and functionality of biological membranes are determined by cooperative interaction between their lipid and protein constituents. Cholesterol is an important structural lipid that modulates fluidity of biological membranes favoring the formation of detergent-resistant microdomains. In the present study, we evaluated the functional role of cholesterol and lipid rafts for entry of hepatitis B viruses into hepatocytes. We show that the duck hepatitis B virus (DHBV) attaches predominantly to detergent-soluble domains on the plasma membrane. Cholesterol depletion from host membranes and thus disruption of rafts does not affect DHBV infection. In contrast, depletion of cholesterol from the envelope of both DHBV and human HBV strongly reduces virus infectivity. Cholesterol depletion increases the density of viral particles and leads to changes in the ultrastructural appearance of the virus envelope. However, the dual topology of the viral envelope protein L is not significantly impaired. Infectivity and density of viral particles are partially restored upon cholesterol replenishment. Binding and entry of cholesterol-deficient DHBV into hepatocytes are not significantly impaired, in contrast to their release from endosomes. We therefore conclude that viral but not host cholesterol is required for endosomal escape of DHBV.     

Caveolin scaffolding region and cholesterol-rich domains in membranes

A protein that constitutes a good marker for a type of cholesterol-rich domain in biological membranes is caveolin. A segment of this protein has a sequence that corresponds to a cholesterol recognition/interaction amino acid consensus (CRAC) motif; this motif has been suggested to cause the incorporation of proteins into cholesterol-rich domains. We have studied the interaction of two peptides containing the CRAC motif of caveolin-1 by differential scanning calorimetry, fluorescence, circular dichroism and magic angle spinning NMR. These peptides promote the segregation of cholesterol into domains from mixtures of the sterol with phosphatidylcholine, as shown by depletion of cholesterol from a portion of the membrane and enrichment of cholesterol in another domain. Cholesterol passes its solubility limit in the cholesterol-rich domain, resulting in the formation of cholesterol crystallites, suggesting that not all of the cholesterol recruited to this domain is bound to the peptide. NMR studies show that the peptides insert somewhat more deeply into membranes when cholesterol is present, but their strongest interaction takes place with the interfacial region of the membrane. We conclude that the peptides we studied containing CRAC sequences are more effective in promoting the formation of cholesterol-rich domains than are shorter peptides of this region of caveolin, which although they contain several aromatic amino acids, they have no CRAC motif. The presence or absence of a CRAC motif, however, is not a sufficient criterion to determine the extent to which a protein will promote the segregation of cholesterol in membranes.