Integrity of the Actin Cytoskeleton of Host Macrophages is Necessary for Mycobacterial Entry.

Macrophages are the primary hosts for Mycobacterium tuberculosis (M. tb), an intracellular pathogen, and the causative organism of tuberculosis (TB) in humans. While M. tb has the ability to enter and survive in host macrophages, the precise mechanism of its internalization, and factors that control this essential process are poorly defined. We have previously demonstrated that perturbations in levels of cholesterol and sphingolipids in macrophages lead to significant reduction in the entry of Mycobacterium smegmatis (M. smegmatis), a surrogate model for mycobacterial internalization, signifying a role for these plasma membrane lipids in interactions at the host-pathogen interface. In this work, we investigated the role of the host actin cytoskeleton, a critical protein framework underlying the plasma membrane, in the entry of M. smegmatis into human macrophages. Our results show that cytochalasin D mediated destabilization of the actin cytoskeleton of host macrophages results in a dose-dependent reduction in the entry of mycobacteria. Notably, the internalization of Escherichia coli remained invariant upon actin destabilization of host cells, implying a specific involvement of the actin cytoskeleton in mycobacterial infection. By monitoring the F-actin content of macrophages utilizing a quantitative confocal microscopy-based technique, we observed a close correlation between the entry of mycobacteria into host macrophages with cellular F-actin content. Our results constitute the first quantitative analysis of the role of the actin cytoskeleton of human macrophages in the entry of mycobacteria, and highlight actin-mediated mycobacterial entry as a potential target for future anti-TB therapeutics.

Structural Stringency and Optimal Nature of Cholesterol Requirement in the Function of the Serotonin1A Receptor.

The role of membrane cholesterol in modulating G protein-coupled receptor (GPCR) structure and function has emerged as a powerful theme in contemporary biology. In this paper, we report the subtlety and stringency involved in the interaction of sterols with the serotonin1A receptor. For this, we utilized two immediate biosynthetic precursors of cholesterol, 7-dehydrocholesterol (7-DHC) and desmosterol, which differ with cholesterol merely in a double bond in their chemical structures in a position-dependent manner. We show that whereas 7-DHC could not support the ligand binding function of the serotonin1A receptor in live cells, desmosterol could partially support it. Importantly, depletion and enrichment of membrane cholesterol over basal level resulted in an increase and reduction of the basal receptor activity, respectively. These results demonstrate the relevance of optimal membrane cholesterol in maintaining the activity of the serotonin1A receptor, thereby elucidating the relevance of cellular cholesterol homeostasis.

Exploring Endocytosis and Intracellular Trafficking of the Human Serotonin1A Receptor.

G protein-coupled receptors (GPCRs) represent the largest class of receptors involved in signal transduction across cell membranes and are major drug targets in all clinical areas. Endocytosis of GPCRs offers a regulatory mechanism for sustaining their signaling within a stringent spatiotemporal regime. In this work, we explored agonist-induced endocytosis of the human serotonin1A receptor stably expressed in HEK-293 cells and the cellular machinery involved in receptor internalization and intracellular trafficking. The serotonin1A receptor is a popular GPCR implicated in neuropsychiatric disorders such as anxiety and depression and serves as an important drug target. In spite of its pharmacological relevance, its mechanism of endocytosis and intracellular trafficking is less understood. In this context, we have utilized a combination of robust population-based flow cytometric analysis and confocal microscopic imaging to address the path and fate of the serotonin1A receptor during endocytosis. Our results, utilizing inhibitors of specific endocytosis pathways and intracellular markers, show that the serotonin1A receptor undergoes endocytosis predominantly via the clathrin-mediated pathway and subsequently recycles to the plasma membrane via recycling endosomes. These results would enhance our understanding of molecular mechanisms of GPCR endocytosis and could offer novel insight into the underlying mechanism of antidepressants that act via the serotonergic pathway. In addition, our results could be relevant in understanding cell (or tissue)-specific GPCR endocytosis.

A Critical Analysis of Molecular Mechanisms Underlying Membrane Cholesterol Sensitivity of GPCRs.

G protein-coupled receptors (GPCRs) are the largest and a diverse family of proteins involved in signal transduction across biological membranes. GPCRs mediate a wide range of physiological processes and have emerged as major targets for the development of novel drug candidates in all clinical areas. Since GPCRs are integral membrane proteins, regulation of their organization, dynamics, and function by membrane lipids, in particular membrane cholesterol, has emerged as an exciting area of research. Cholesterol sensitivity of GPCRs could be due to direct interaction of cholesterol with the receptor (specific effect). Alternately, GPCR function could be influenced by the effect of cholesterol on membrane physical properties (general effect). In this review, we critically analyze the specific and general mechanisms of the modulation of GPCR function by membrane cholesterol, taking examples from representative GPCRs. While evidence for both the proposed mechanisms exists, there appears to be no clear-cut distinction between these two mechanisms, and a combination of these mechanisms cannot be ruled out in many cases. We conclude that classifying the mechanism underlying cholesterol sensitivity of GPCR function merely into these two mutually exclusive classes could be somewhat arbitrary. A more holistic approach could be suitable for analyzing GPCR-cholesterol interaction.

Exploring oligomeric state of the serotonin1A receptor utilizing photobleaching image correlation spectroscopy: implications for receptor function.

The oligomerization of G protein-coupled receptors (GPCRs) represents an important process in GPCR function and drug discovery. We have addressed cholesterol-dependent oligomerization state of the serotonin1A receptor, a representative GPCR and an important drug target, utilizing photobleaching image correlation spectroscopy (pbICS). pbICS allows determination of oligomeric state of membrane receptors since change in cluster density upon photobleaching is dependent on the oligomeric state. Our results show that oligomeric state of the serotonin1A receptor is modulated by cell membrane cholesterol and a trimeric population of the receptor prevails in control (normal) cholesterol conditions. Interestingly, upon lowering membrane cholesterol, the predominant oligomeric population of the receptor changes to dimers. This is associated with an increase in specific ligand binding activity of the receptor, thereby implying a crucial role of receptor dimers in ligand binding activity. Upon cholesterol replenishment, the distribution of receptor oligomers is further changed such that the trimers become the major population, with a concomitant restoration of ligand binding activity to the control level. These results demonstrate the utility of pbICS in monitoring oligomeric states of membrane receptors in general, and the cholesterol-dependent oligomeric state of the serotonin1A receptor in particular. We envision that functional correlates of oligomeric states of GPCRs could provide better understanding of GPCR function in health and disease, and help design better therapeutic strategies.

Identification of Sphingolipid-binding Motif in G Protein-coupled Receptors.

Sphingolipids correspond to a major class of lipids which serve as indispensable structural components of membranes and play an important role in various cellular functions. They constitute ~10-20% of total membrane lipids and are known to form segregated domains in biological membranes. Sphingolipids have been shown to play a vital role in the function of various G protein-coupled receptors (GPCRs). We report here the presence of sphingolipid-binding motif (SBM) in representative GPCRs such as cholecystokinin, oxytocin and secretin receptors, and subtypes of human serotonin receptors. We previously reported the importance of sphingolipids in the function of the serotonin1A receptor, a representative member of the GPCR superfamily, involved in behavioral, cognitive, and developmental functions. In this work, we show that the serotonin1A receptor contains a putative SBM, corresponding to amino acids 205 to 213. In addition, our analysis shows that SBM is an intrinsic characteristic feature of the serotonin1A receptor and is conserved throughout the course of natural evolution. Our results represent the first report on the presence of SBM in serotonin1A receptors and provide novel insight on the molecular mechanism of GPCR-sphingolipid interaction.

Statin-induced chronic cholesterol depletion inhibits Leishmania donovani infection: Relevance of optimum host membrane cholesterol.

Leishmania are obligate intracellular protozoan parasites that invade and survive within host macrophages leading to leishmaniasis, a major cause of mortality and morbidity worldwide, particularly among economically weaker sections in tropical and subtropical regions. Visceral leishmaniasis is a potent disease caused by Leishmania donovani. The detailed mechanism of internalization of Leishmania is poorly understood. A basic step in the entry of Leishmania involves interaction of the parasite with the host plasma membrane. In this work, we have explored the effect of chronic metabolic cholesterol depletion using lovastatin on the entry and survival of Leishmania donovani in host macrophages. We show here that chronic cholesterol depletion of host macrophages results in reduction in the attachment of Leishmania promastigotes, along with a concomitant reduction in the intracellular amastigote load. These results assume further relevance since chronic cholesterol depletion is believed to mimic physiological cholesterol modulation. Interestingly, the reduction in the ability of Leishmania to enter host macrophages could be reversed upon metabolic replenishment of cholesterol. Importantly, enrichment of host membrane cholesterol resulted in reduction in the entry and survival of Leishmania in host macrophages. As a control, the binding of Escherichia coli to host macrophages remained invariant under these conditions, thereby implying specificity of cholesterol requirement for effective leishmanial infection. To the best of our knowledge, these results constitute the first comprehensive demonstration that an optimum content of host membrane cholesterol is necessary for leishmanial infection. Our results assume relevance in the context of developing novel therapeutic strategies targeting cholesterol-mediated leishmanial infection.

The ganglioside GM1 interacts with the serotonin1A receptor via the sphingolipid binding domain.

Glycosphingolipids are minor yet essential components of eukaryotic cell membranes and are involved in a variety of cellular processes. Although glycosphingolipids such as GM1 have been previously reported to influence the function of G protein-coupled receptors (GPCRs), the molecular mechanism remains elusive. In this paper, we have explored the interaction of GM1 with the serotonin1A receptor, an important neurotransmitter receptor that belongs to the GPCR family. To examine the molecular basis of the interaction of GM1 with the serotonin1A receptor, we performed a series of coarse-grain molecular dynamics simulations of the receptor embedded in membrane bilayers containing GM1. Our results show that GM1 interacts with the serotonin1A receptor predominantly at the extracellular loop 1 and specifically at the sphingolipid binding domain (SBD). The SBD motif consists of a characteristic combination of aromatic, basic and turn-inducing residues, and is evolutionarily conserved in case of the serotonin1A receptor. The interaction of the SBD site with GM1 appears to stabilize a 'flip-out' conformation in which W102 of the extracellular loop 1 flips out from the central lumen of the receptor toward the membrane. The population of the 'flip-out' conformation is increased in the presence of cholesterol. Our data strongly suggest that a direct interaction between GM1 and the SBD site of the serotonin1A receptor may occur in vivo. In view of the reported role of GM1 and the serotonin1A receptor in neurodegenerative diseases, GM1-receptor interaction assumes significance in the context of malfunctioning of neuronal GPCRs under such conditions.

Cholesterol modulates bitter taste receptor function.

Bitter taste perception in humans is believed to act as a defense mechanism against ingestion of potential toxic substances. Bitter taste is perceived by 25 distinct bitter taste receptors (T2Rs) which belong to the family of G protein-coupled receptors (GPCRs). In the overall context of the role of membrane lipids in GPCR function, we show here that T2R4, a representative member of the bitter taste receptor family, displays cholesterol sensitivity in its signaling function. In order to gain further insight into cholesterol sensitivity of T2R4, we mutated two residues Tyr114(3.59) and Lys117(3.62) present in the cholesterol recognition amino acid consensus (CRAC) motif in T2R4 with alanines. We carried out functional characterization of the mutants by calcium mobilization, followed by cholesterol depletion and replenishment. CRAC motifs in GPCRs have previously been implicated in preferential cholesterol association. Our analysis shows that the CRAC motif represents an intrinsic feature of bitter taste receptors and is conserved in 22 out of 25 human T2Rs. We further demonstrate that Lys117, an important CRAC residue, is crucial in the reported cholesterol sensitivity of T2R4. Interestingly, cholesterol sensitivity of T2R4 was observed at quinine concentrations in the lower mM range. To the best of our knowledge, our results represent the first report addressing the molecular basis of cholesterol sensitivity in the function of taste receptors.

Sphingolipids modulate the function of human serotonin1A receptors: Insights from sphingolipid-deficient cells.

Sphingolipids are essential components of eukaryotic cell membranes and are known to modulate a variety of cellular functions. It is becoming increasingly clear that membrane lipids play a crucial role in modulating the function of integral membrane proteins such as G protein-coupled receptors (GPCRs). In this work, we utilized LY-B cells, that are sphingolipid-auxotrophic mutants defective in sphingolipid biosynthesis, to monitor the role of cellular sphingolipids in the function of an important neurotransmitter receptor, the serotonin1A receptor. Serotonin1A receptors belong to the family of GPCRs and are implicated in behavior, development and cognition. Our results show that specific ligand binding and G-protein coupling of the serotonin1A receptor exhibit significant enhancement under sphingolipid-depleted conditions, which reversed to control levels upon replenishment of cellular sphingolipids. In view of the reported role of sphingolipids in neuronal metabolism and pathogenesis of several neuropsychiatric disorders, exploring the role of serotonin1A receptors under conditions of defective sphingolipid metabolism assumes relevance, and could contribute to our overall understanding of such neuropsychiatric disorders. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.

Integrity of the actin cytoskeleton of host macrophages is essential for Leishmania donovani infection.

Visceral leishmaniasis is a vector-borne disease caused by an obligate intracellular protozoan parasite Leishmania donovani. The molecular mechanism involved in internalization of Leishmania is poorly understood. The entry of Leishmania involves interaction with the plasma membrane of host cells. We have previously demonstrated the requirement of host membrane cholesterol in the binding and internalization of L. donovani into macrophages. In the present work, we explored the role of the host actin cytoskeleton in leishmanial infection. We observed a dose-dependent reduction in the attachment of Leishmania promastigotes to host macrophages upon destabilization of the actin cytoskeleton by cytochalasin D. This is accompanied by a concomitant reduction in the intracellular amastigote load. We utilized a recently developed high resolution microscopy-based method to quantitate cellular F-actin content upon treatment with cytochalasin D. A striking feature of our results is that binding of Leishmania promastigotes and intracellular amastigote load show close correlation with cellular F-actin level. Importantly, the binding of Escherichia coli remained invariant upon actin destabilization of host cells, thereby implying specific involvement of the actin cytoskeleton in Leishmania infection. To the best of our knowledge, these novel results constitute the first comprehensive demonstration on the specific role of the host actin cytoskeleton in Leishmania infection. Our results could be significant in developing future therapeutic strategies to tackle leishmaniasis.

Stereospecific requirement of cholesterol in the function of the serotonin1A receptor.

The serotonin1A receptor is an important member of the G protein-coupled receptor (GPCR) family. It is involved in the generation and modulation of a variety of cognitive and behavioral functions and serves as a drug target. Previous work from our laboratory has established the sensitivity of the function of the serotonin1A receptor to membrane cholesterol. Solubilization of the hippocampal serotonin1A receptor utilizing the zwitterionic detergent CHAPS is accompanied by loss of cholesterol and results in reduction in specific ligand binding. Replenishment of cholesterol to solubilized membranes restores specific ligand binding to the receptor. We utilized this strategy of sterol replenishment of solubilized membranes to explore the stereospecific stringency of cholesterol for receptor function. We used two stereoisomers of cholesterol, ent-cholesterol (enantiomer of cholesterol) and epi-cholesterol (a diastereomer of cholesterol), for this purpose. Importantly, we show here that while ent-cholesterol could replace cholesterol in supporting receptor function, epi-cholesterol could not. These results imply that the requirement of membrane cholesterol for the serotonin1A receptor function is diastereospecific, yet not enantiospecific. Our results extend and help define specificity of the interaction of membrane cholesterol with the serotonin1A receptor, and represent the first report utilizing ent-cholesterol to examine stereospecificity of GPCR-cholesterol interaction.

Desmosterol replaces cholesterol for ligand binding function of the serotonin(1A) receptor in solubilized hippocampal membranes: support for nonannular binding sites for cholesterol?

The serotonin(1A) receptor is an important member of the G-protein coupled receptor family, and is involved in the generation and modulation of a variety of cognitive and behavioral functions. Solubilization of the hippocampal serotonin(1A) receptor by CHAPS is accompanied by loss of cholesterol that results in a reduction in specific agonist binding activity. Replenishment of cholesterol to solubilized membranes restores membrane cholesterol content and significantly recovers specific agonist binding. In order to test the stringency of cholesterol requirement, we solubilized native hippocampal membranes followed by replenishment with desmosterol. Desmosterol is the immediate biosynthetic precursor of cholesterol in the Bloch pathway differing only in a double bond at the 24th position. Our results show that replenishment with desmosterol restores ligand binding of serotonin(1A) receptors. This is consistent with earlier results showing that desmosterol can replace cholesterol in a large number of cases. However, these results appear to be contradictory to our earlier findings, performed by sterol manipulation utilizing methyl-ß-cyclodextrin, in which we observed that replacing cholesterol with desmosterol is unable to restore specific ligand binding of the hippocampal serotonin(1A) receptor. We discuss the possible molecular mechanism, in terms of nonannular lipid binding sites around the receptor, giving rise to these differences.

A novel mechanism for an old drug: amphotericin B in the treatment of visceral leishmaniasis.

Visceral leishmaniasis (VL) is caused by various species of the genus Leishmania. Internalization of Leishmania into host cells is facilitated by a large number of receptors, and therefore no panacea is available for the treatment of leishmaniasis. We previously demonstrated the requirement of host membrane cholesterol in the entry of Leishmania into macrophages by cholesterol depletion using methyl-ß-cyclodextrin (MßCD). We recently showed that leishmanial infection is inhibited upon sequestration of host membrane cholesterol using amphotericin B (AmB), considered as the best existing drug against VL. The reason for the antileishmanial activity of AmB is generally believed to be its ability to bind ergosterol in parasite membranes. Our recent results offer the opportunity to reexamine the mechanism behind the effectiveness of current AmB-based therapeutic strategies to treat leishmaniasis. We propose here a novel mechanism in which the effectiveness of AmB treatment could be partly based on its ability to sequester cholesterol in the host membrane, thereby abrogating macrophage-parasite interaction.

Identification of cholesterol recognition amino acid consensus (CRAC) motif in G-protein coupled receptors.

G-protein coupled receptors (GPCRs) are the largest class of molecules involved in signal transduction across membranes, and represent major targets in the development of novel drug candidates in all clinical areas. Membrane cholesterol has been reported to have an important role in the function of a number of GPCRs. Several structural features of proteins, believed to result in preferential association with cholesterol, have been recognized. Cholesterol recognition/interaction amino acid consensus (CRAC) sequence represents such a motif. Many proteins that interact with cholesterol have been shown to contain the CRAC motif in their sequence. We report here the presence of CRAC motifs in three representative GPCRs, namely, rhodopsin, the ß(2)-adrenergic receptor, and the serotonin(1A) receptor. Interestingly, the function of these GPCRs has been previously shown to be dependent on membrane cholesterol. The presence of CRAC motifs in GPCRs indicates that interaction of cholesterol with GPCRs could be specific in nature. Further analysis shows that CRAC motifs are inherent characteristic features of the serotonin(1A) receptor and are conserved over natural evolution. These results constitute the first report of the presence of CRAC motifs in GPCRs and provide novel insight in the molecular nature of GPCR-cholesterol interaction.

A molecular sensor for cholesterol in the human serotonin1A receptor.

The function of several G protein-coupled receptors (GPCRs) exhibits cholesterol sensitivity. Cholesterol sensitivity of GPCRs could be attributed to specific sequence and structural features, such as the cholesterol recognition/interaction amino acid consensus (CRAC) motif, that facilitate their cholesterol-receptor interaction. In this work, we explored the molecular basis of cholesterol sensitivity exhibited by the serotonin1A receptor, the most studied GPCR in the context of cholesterol sensitivity, by generating mutants of key residues in CRAC motifs in transmembrane helix 2 (TM2) and TM5 of the receptor. Our results show that a lysine residue (K101) in one of the CRAC motifs is crucial for sensing altered membrane cholesterol levels. Insights from all-atom molecular dynamics simulations showed that cholesterol-sensitive functional states of the serotonin1A receptor are associated with reduced conformational dynamics of extracellular loops of the receptor. These results constitute one of the first reports on the molecular mechanism underlying cholesterol sensitivity of GPCRs.

Differential effects of cholesterol and desmosterol on the ligand binding function of the hippocampal serotonin(1A) receptor: implications in desmosterolosis.

Cholesterol is a unique molecule in terms of high level of in-built stringency, fine tuned by natural evolution for its ability to optimize physical properties of higher eukaryotic cell membranes in relation to biological functions. We previously demonstrated the requirement of membrane cholesterol in maintaining the ligand binding activity of the hippocampal serotonin(1A) receptor. In order to test the molecular stringency of the requirement of cholesterol, we depleted cholesterol from native hippocampal membranes followed by replenishment with desmosterol. Desmosterol is an immediate biosynthetic precursor of cholesterol in the Bloch pathway differing only in a double bond at the 24th position in the alkyl side chain. Our results show that replenishment with desmosterol does not restore ligand binding activity of the serotonin(1A) receptor although replenishment with cholesterol led to significant recovery of ligand binding. This is in spite of similar membrane organization (order) in these membranes, as monitored by fluorescence anisotropy measurements. The requirement for restoration of ligand binding activity therefore appears to be more stringent than the requirement for the recovery of overall membrane order. These novel results have potential implications in understanding the interaction of membrane lipids with this important neuronal receptor in diseases such as desmosterolosis.

Identification of a red-emitting fluorescent ligand for in vitro visualization of human serotonin 5-HT(1A) receptors.

The 5-HT(1A) receptor subtype is the most thoroughly studied serotonin receptor subtype. We report here the design, synthesis and characterization of two new fluorescent ligands for the 5-HT(1A) receptor. The new 1-arylpiperazine-based red-emitting fluorescent compound 6 displayed good binding affinity at the 5-HT(1A) receptor (K(i)=35 nM) and was able to label specifically the human 5-HT(1A) receptor stably expressed in CHO cells visualized using confocal laser scanning microscopy.

Effect of sphingomyelinase treatment on ligand binding activity of human serotonin1A receptors.

The serotonin1A receptor is an important member of the G-protein coupled receptor family, and is involved in the generation and modulation of a variety of cognitive, behavioral, and developmental functions. We have monitored the ligand binding of the human serotonin1A receptor stably expressed in CHO cells (termed CHO-5-HT1AR) following treatment with sphingomyelinase (SMase), an enzyme that specifically catalyzes the hydrolysis of sphingomyelin into ceramide and phosphorylcholine. Our results show, for the first time, that the specific ligand binding activity of the serotonin1A receptor in membranes isolated from CHO-5-HT1AR cells is increased upon sphingomyelinase treatment. Saturation binding analysis reveals increase in binding affinity of the receptor under these conditions. This is accompanied by a reduction in membrane order, as monitored by fluorescence anisotropy of the membrane probe 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) in intact cells. These results represent the first report on the effect of sphingomyelinase treatment on the ligand binding activity of this important neurotransmitter receptor.