NMR conformational analyses on (des-bromo) neuropeptide B [1-23] and neuropeptide W [1-23]: the importance of alpha-helices, a cation-pi interaction and a beta-turn

The preferred conformations of the orphan G-protein coupled receptor agonists (des-bromo) neuropeptide B [1-23] and neuropeptide W [1-23], referred to as NPB and NPW, have been determined by (1)H NMR, CD, and molecular modeling. The sequences of NPB and NPW are WYKPAAGHSSYSVGRAAGLLSGL and WYKHVASPRYHTVGRAAGLLMGL, respectively. These are hypothalamic peptides that exert their biological actions on GPR7 and GPR8 receptors. Micellar solutions using the membrane mimetic, sodium dodecylsulphate-d(25) (SDS), were used to mimic a physiological environment for the peptides. The secondary structure of NPB consists of a type II beta-turn involving residues Lys(3) to Ala(6). The C-terminal region of NPB exists in a conformational equilibrium between different secondary structures, including an alpha-helix from residues Arg(15) to Ser(21), and a 3(10)-helix from residues Ser(12) to Ser(21). The N-terminus of NPW exhibits a cation-pi interaction between the Lys(3) side chain and the quadrupole moment of the Trp(1) indole group. At the C-terminus of NPW, a well-defined alpha-helical conformation exists from Arg(15) to Met(21). As NPB and NPW have 91% sequence homology from residues Val(13) to Leu(23), with only residue 21 differing between the two peptides, the similar C-terminal secondary structures of these two peptides are consistent with the sequences. This is supported by the similar CD spectra. The different secondary structures at the N-termini for NPB and NPW point to the importance of the N-terminus in receptor binding. This is consistent with the work of Fujii et al. [J. Biol. Chem. 277, 34010-34016 (2002)] who observed that iodination of the NPB Tyr(2) resulted in decreased agonistic activity at GPR7. In addition, Tanaka et al. [Proc. Natl. Acad. Sci. USA 100, 6251-6256 (2003)] showed that deletion of Trp(1) from NPB or NPW drastically decreased activity at GPR7 for NPB and GPR7 and GPR8 for NPW. Therefore, we postulate that the N-terminus is involved in membrane recognition and receptor binding.     

Identification of neuropeptide W as the endogenous ligand for orphan G-protein-coupled receptors GPR7 and GPR8

The structurally related orphan G-protein-coupled receptors GPR7 and GPR8 are expressed in the central nervous system, and their ligands have not been identified. Here, we report the identification of the endogenous ligand for both of these receptors. We purified the peptide ligand from porcine hypothalamus using stable Chinese hamster ovary cell lines expressing human GPR8 and cloned the cDNA encoding its precursor protein. The cDNA encodes two forms of the peptide ligand with lengths of 23 and 30 amino acid residues as mature peptides. We designated the two ligands neuropeptide W-23 (NPW23) and neuropeptide W-30 (NPW30). The amino acid sequence of NPW23 is completely identical to that of the N-terminal 23 residues of NPW30. Synthetic NPW23 and NPW30 activated and bound to both GPR7 and GPR8 at similar effective doses. Intracerebroventricular administration of NPW23 in rats increased food intake and stimulated prolactin release. These findings indicate that neuropeptide W is the endogenous ligand for both GPR7 and GPR8 and acts as a mediator of the central control of feeding and the neuroendocrine system.     

Development of a potent and selective GPR7 (NPBW1) agonist: a systematic structure-activity study of neuropeptide B.

Neuropeptide B (NPB) has been recently identified as an endogenous ligand for GPR7 (NPBW1) and GPR8 (NPBW2) and has been shown to possess a relatively high selectivity for GPR7. In order to identify useful experimental tools to address physiological roles of GPR7, we synthesized a series of NPB analogs based on modification of an unbrominated form of 23 amino acids with amidated C-terminal, Br(-)NPB-23-NH(2). We confirmed that truncation of the N-terminal Trp residue resulted in almost complete loss of the binding affinity of NPB for GPR7 and GPR8, supporting the special importance of this residue for binding. Br(-)NPB-23-NH2 analogs in which each amino acid in positions 4, 5, 7, 8, 9, 10, 12 and 21 was replaced with alanine or glycine exhibited potent binding affinity comparable to the parent peptide. In contrast, replacement of Tyr(11) with alanine reduced the binding affinity for both GPR7 and GPR8 four fold. Of particular interest, several NPB analogs in which the consecutive amino acids from Pro4 to Val(13) were replaced with several units of 5-aminovaleric acid (Ava) linkers retained their potent affinity for GPR7. Furthermore, these Ava-substituted NPB analogs exhibited potent agonistic activities for GPR7 expressed in HEK293 cells. Among the Ava-substituted NPB analogs, analog 15 (Ava-5) and 17 (Ava-3) exhibited potency comparable to the parent peptide for GPR7 with significantly reduced activity for GPR8, resulting in high selectivity for GPR7. These highly potent and selective NPB analogs may be useful pharmacological tools to investigate the physiological and pharmacological roles of GPR7.     

Neuropeptide B and W regulate leptin and resistin secretion, and stimulate lipolysis in isolated rat adipocytes

Neuropeptide B (NPB) and W (NPW) regulate food intake and energy homeostasis in humans via two G-protein-coupled receptor subtypes, termed as GPR7 and GPR8. Rodents express GPR7 only. In animals, NPW decreases insulin and leptin levels, whereas the deletion of either NPB or GPR7 leads to obesity and hyperphagia. Metabolic and endocrine in vitro activities of NPW/NPB in adipocytes are unknown. We therefore characterize the effects of NPB and NPW on the secretion and expression of leptin and resistin, and on lipolysis, using rat adipocytes. Isolated rat adipocytes express GPR7 mRNA. NPB and NPW are expressed in macrophages and preadipocytes but are absent in mature adipocytes. Both, NPB and NPW reduce the secretion and expression of leptin from isolated rat adipocytes. NPB stimulates the secretion and expression of resistin, whereas both, NPB and NPW increase lipolysis. Our study demonstrates for the first time that NPB and NPW regulate the expression and secretion of leptin and resistin, and increase lipolysis in isolated rat adipocytes. These effects are presumably mediated via GPR7. The increase of resistin secretion, stimulation of lipolysis and the decrease of leptin secretion may represent mechanisms, through which NPB and NPW can affect glucose and lipid homeostasis, and food intake in rodents.     

Expression of neuropeptide W in rat stomach mucosa: regulation by nutritional status, glucocorticoids and thyroid hormones

Neuropeptide W (NPW) is a recently identified neuropeptide that binds to G-protein-coupled receptor 7 (GPR7) and 8 (GPR8). In rodent brain, NPW mRNA is confined to specific nuclei in hypothalamus, midbrain and brainstem. Expression of NPW mRNA has also been confirmed in peripheral organs such as stomach. Several reports suggested that brain NPW is implicated in the regulation of energy and hormonal homeostasis, namely the adrenal and thyroid axes; however the precise physiological role and regulation of peripheral NPW remains unclear. In this study, we examined the effects of nutritional status on the regulation of NPW in stomach mucosa. Our results show that in this tissue, NPW mRNA and protein expression is negatively regulated by fasting and food restriction, in all the models we studied: males, females and pregnant females. Next, we examined the effect of glucocorticoids and thyroid hormones on NPW mRNA expression in the stomach mucosa. Our data showed that NPW expression is decreased in this tissue after glucocorticoid treatment or hyperthyroidism. Conversely, hypothyroidism induces a marked increase in the expression of NPW in rat stomach. Overall, these data indicate that stomach NPW is regulated by nutritional and hormonal status.     

Cardiovascular actions of central neuropeptide W in conscious rats

Neuropeptide W (NPW) is a novel hypothalamic peptide that activates the orphan G protein-coupled receptors, GPR7 and GPR8. Two endogenous molecular forms of NPW that consist of 23- and 30-amino acid residues were identified. Intracerebroventricular (i.c.v.) administration of NPW is known to suppress spontaneous-feeding at dark-phase and fasting-induced food intake and to decrease body weight and plasma growth hormone and to increase prolactin and corticosterone; however, little is known about its effect on other physiological functions. We examined the effects of i.c.v. administration of NPW30 (0.3 and 3 nmol) on the mean arterial pressure (MAP), heart rate (HR), and plasma norepinephrine and epinephrine in conscious rats. NPW30 (3 nmol) provoked increases in MAP (85.12+/-3.16 to 106.26+/-2.66 mm Hg) and HR (305.75+/-13.76 to 428.45+/-26.82 beats/min) and plasma norepinephrine (138.1+/-18.1 to 297.2+/-25.9 pg/ml) and epinephrine (194.6+/-21.4 to 274.6+/-22.7 pg/ml). Intravenously administered NPW30 (3 nmol) had no significant effects on MAP and HR. These results indicate that central NPW30 increases sympathetic nervous outflow and affects cardiovascular function.     

Neuronal interactions between neuropeptide W- and orexin- or melanin-concentrating hormone-containing neurons in the rat hypothalamus

Neuropeptide W (NPW) was recently discovered as the endogenous ligand for GPR7 and GPR8, which are orphan G protein-coupled receptors isolated from the porcine brain. These receptors are assumed to be involved in feeding regulation and/or energy homeostasis. Recent anatomical studies have revealed that high levels of GPR7 mRNA are distributed in the brain, including the hypothalamus and amygdala. However immunohistochemical studies on the distribution and localization of NPW have revealed differing results concerning whether or not NPW-containing cell bodies and their processes are present in the hypothalamus. Only a few immunohistochemical reports have been published concerning the presence of NPW-containing neurons in the brains of rodents, while there have been no anatomical studies of the co-localization of this neuropeptide with other transmitters. On this basis, we used a specific antiserum against NPW to determine immunohistochemically the presence of NPW-containing neurons in the rat hypothalamus. Many NPW-like immunoreactive cell bodies and their processes could be detected in the caudal region of the lateral hypothalamus but not in its anterior or middle regions. Given this positive identification of NPW-containing neurons in the lateral hypothalamus, we further studied the nature of interaction between NPW-containing neurons and neurons containing feeding regulating peptides such as orexin- and melanin-concentrating hormone (MCH). Very close interactions between NPW-containing nerve processes and orexin- and MCH-containing neuronal cell bodies and processes could be observed. These morphological findings strongly suggest that NPW is involved in the regulation of feeding and/or sleep/arousal behavior through orexin- and/or MCH-mediated neuronal pathways.     

A chronic high fat diet alters the homologous and heterologous control of appetite regulating peptide receptor expression

Leptin, ghrelin and neuropeptide W (NPW) modulate vagal afferent activity, which may underlie their appetite regulatory actions. High fat diet (HFD)-induced obesity induces changes in the plasma levels of these peptides and alters the expression of receptors on vagal afferents. We investigated homologous and heterologous receptor regulation by leptin, ghrelin and NPW. Mice were fed (12 weeks) a standard laboratory diet (SLD) or HFD. Nodose ganglia were cultured overnight in the presence or absence of each peptide. Leptin (LepR), ghrelin (GHS-R), NPW (GPR7) and cholecystokinin type-1 (CCK1R) receptor mRNA, and the plasma leptin, ghrelin and NPW levels were measured. SLD: leptin reduced LepR, GPR7, increased GHS-R and CCK1R mRNA; ghrelin increased LepR, GPR7, CCK1R, and decreased GHS-R. HFD: leptin decreased GHS-R and GPR7, ghrelin increased GHS-R and GPR7. NPW decreased all receptors except GPR7 which increased with HFD. Plasma leptin was higher and NPW lower in HFD. Thus, HFD-induced obesity disrupts inter-regulation of appetite regulatory receptors in vagal afferents.     

Injection of neuropeptide W into paraventricular nucleus of hypothalamus increases food intake

Neuropeptide W (NPW) is an endogenous ligand for G protein-coupled receptor 7 (GPR7). There are two forms of the peptide, designated as neuropeptide W-23 (NPW23) and neuropeptide W-30 (NPW30). In the current study we found that intracerebroventricular administration of NPW23 increased c-Fos immunoreactivity (IR) in a variety of brain sites, many of which are involved in the regulation of feeding. In particular, we noted that c-Fos IR levels were increased in hypocretin-expressing neurons in the perifornical region of the lateral hypothalamus (LH). We then studied whether injection of NPW23 into the paraventricular nucleus of the hypothalamus (PVN) and the LH increased food intake over a 24-h time period. Intra-PVN injection of NPW23 at doses ranging from 0.1 to 3 nmol increased feeding for up to 4 h, and doses ranging from 0.3 to 3 nmol increased feeding for up to 24 h. In contrast, only the 3-nmol dose of NPW23 increased feeding after administration into the LH. Together, these data suggest a modulatory role for NPW in the control of food intake.     

Neuropeptide W regulates proliferation and differentiation of ATDC5: Possible involvement of GPR7 activation,PKA and PKC‐dependent signalling cascades

Various neuropeptides related to the energy equilibrium affect bone growth in humans and animals. Neuropeptides W (NPW) are identical in the internal ligands of the two G‐protein receptors (GPRs) included in subtypes 7 and 8. Neuropeptides W inhibits proliferation in the cultivated rat calvarial osteoblast‐like (ROB) cells. This study examines the expression of NPW and GPR7 in murine chondrocyte and their function. An immunohistochemical analysis showed that NPW and GPR7 were expressed in the proliferative chondrocytes of the growth plates in the hind limbs of mice. The NPW mRNA quickly elevated in the early differentiation (7‐14 days) of ATDC5 cells, while NPW and GPR7 mRNA were reduced during the late stage (14‐21 days) of differentiation. Neuropeptide W‐23 (NPW‐23) promoted the proliferation of ATDC5 cells, which was attenuated by inhibiting the GPR7, protein kinase A (PKA), protein kinase C (PKC) and ERK1/2 pathways. Neuropeptide W‐23 enhanced the early cell differentiation, as evaluated by collagen type II and the aggrecan gene expression, which was unaffected by inhibiting the ERK1/2 pathway, but significantly decreased by inhibiting the PKA, PKC and p38 MAPK pathways. In contrast, NPW‐23 was not involved in the terminal differentiation of the chondrocytes, as evaluated by the mineralization of the chondrocytes and the activity of the alkaline phosphatase. Neuropeptides W stimulated the PKA, PKC, p38 MAPK and ERK1/2 activities in a dose‐ and time‐dependent manner in the ATDC5 cells. These results show that NPW promotes the proliferation and early differentiation of murine chondrocyte via GPR7 activation, as well as PKA and PKC‐dependent signalling cascades, which may be involved in endochondral bone formation.     

Ontogeny of a new enteric peptide, neuropeptide W (NPW), in the developing rat stomach

Neuropeptide W (NPW), a novel endogenous peptide for G protein-coupled receptors GPR7 and GPR8, is expressed in the gastric antral mucosa of rat, mouse, and human stomachs. Here, we studied the ontogeny of NPW in the developing rat stomach. Real-time RT-PCR showed that NPW gene expression was initially detectable in embryonic day 14 (E14) stomach and gradually increased during the progress of age until birth, postnatal day 1 (P1). NPW mRNA level in the stomach increased again from the weaning period (P21) until reaching adulthood. Immunohistochemistry using polyclonal antibodies raised against rat NPW revealed that NPW-positive cells were detected in the P1 antral stomach and gradually increased during the development of age. Furthermore, double immunohistochemistry demonstrated that NPW colocalized with gastrin in P1 rat stomach. These data will provide clues to physiological functions of NPW in the development of rat stomach.     

Deciphering the GPER/GPR30-agonist and antagonists interactions using molecular modeling studies,molecular dynamics,and docking simulations

The G-protein coupled estrogen receptor 1 GPER/GPR30 is a transmembrane seven-helix (7TM) receptor involved in the growth and proliferation of breast cancer. Due to the absence of a crystal structure of GPER/GPR30, in this work, molecular modeling studies have been carried out to build a three-dimensional structure, which was subsequently refined by molecular dynamics (MD) simulations (up to 120 ns). Furthermore, we explored GPER/GPR30’s molecular recognition properties by using reported agonist ligands (G1, estradiol (E2), tamoxifen, and fulvestrant) and the antagonist ligands (G15 and G36) in subsequent docking studies. Our results identified the E2 binding site on GPER/GPR30, as well as other receptor cavities for accepting large volume ligands, through GPER/GPR30 π–π, hydrophobic, and hydrogen bond interactions. Snapshots of the MD trajectory at 14 and 70 ns showed almost identical binding motifs for G1 and G15. It was also observed that C107 interacts with the acetyl oxygen of G1 (at 14 ns) and that at 70 ns the residue E275 interacts with the acetyl group and with the oxygen from the other agonist whereas the isopropyl group of G36 is oriented toward Met141, suggesting that both C107 and E275 could be involved in the protein activation. This contribution suggest that GPER1 has great structural changes which explain its great capacity to accept diverse ligands, and also, the same ligand could be recognized in different binding pose according to GPER structural conformations.     

Identification of a neuropeptide modified with bromine as an endogenous ligand for GPR7

We isolated a novel gene in a search of the Celera data base and found that it encoded a peptidic ligand for a G protein-coupled receptor, GPR7 (O'Dowd, B. F., Scheideler, M. A., Nguyen, T., Cheng, R., Rasmussen, J. S., Marchese, A., Zastawny, R., Heng, H. H., Tsui, L. C., Shi, X., Asa, S., Puy, L., and George, S. R. (1995) Genomics 28, 84-91; Lee, D. K., Nguyen, T., Porter, C. A., Cheng, R., George, S. R., and O'Dowd, B. F. (1999) Mol. Brain Res. 71, 96-103). The expression of this gene was detected in various tissues in rats, including the lymphoid organs, central nervous system, mammary glands, and uterus. GPR7 mRNA was mainly detected in the central nervous system and uterus. In situ hybridization showed that the gene encoding the GPR7 ligand was expressed in the hypothalamus and hippocampus of rats. To determine the molecular structure of the endogenous GPR7 ligand, we purified it from bovine hypothalamic tissue extracts on the basis of cAMP production-inhibitory activity to cells expressing GPR7. Through structural analyses, we found that the purified endogenous ligand was a peptide with 29 amino acid residues and that it was uniquely modified with bromine. We subsequently determined that the C-6 position of the indole moiety in the N-terminal Trp was brominated. We believe this is the first report on a neuropeptide modified with bromine and have hence named it neuropeptide B. In in vitro assays, bromination did not influence the binding of neuropeptide B to the receptor.     

Conformation of alamethicin in oriented phospholipid bilayers determined by (15)N solid-state nuclear magnetic resonance

The conformation of the 20-residue antibiotic ionophore alamethicin in macroscopically oriented phospholipid bilayers has been studied using (15)N solid-state nuclear magnetic resonance (NMR) spectroscopy in combination with molecular modeling and molecular dynamics simulations. Differently (15)N-labeled variants of alamethicin and an analog with three of the alpha-amino-isobutyric acid residues replaced by alanines have been investigated to establish experimental structural constraints and determine the orientation of alamethicin in hydrated phospholipid (dimyristoylphosphatidylcholine) bilayers and to investigate the potential for a major kink in the region of the central Pro(14) residue. From the anisotropic (15)N chemical shifts and (1)H-(15)N dipolar couplings determined for alamethicin with (15)N-labeling on the Ala(6), Val(9), and Val(15) residues and incorporated into phospholipid bilayer with a peptide:lipid molar ratio of 1:8, we deduce that alamethicin has a largely linear alpha-helical structure spanning the membrane with the molecular axis tilted by 10-20 degrees relative to the bilayer normal. In particular, we find compatibility with a straight alpha-helix tilted by 17 degrees and a slightly kinked molecular dynamics structure tilted by 11 degrees relative to the bilayer normal. In contrast, the structural constraints derived by solid-state NMR appear not to be compatible with any of several model structures crossing the membrane with vanishing tilt angle or the earlier reported x-ray diffraction structure (Fox and Richards, Nature. 300:325-330, 1982). The solid-state NMR-compatible structures may support the formation of a left-handed and parallel multimeric ion channel.     

Role of C-terminal Membrane-proximal Basic Residues in Cell Surface Trafficking of HIV Coreceptor GPR15 Protein

Cell surface density of G protein-coupled receptors (GPCRs) is controlled by dynamic molecular interactions that often involve recognition of the distinct sequence signals on the cargo receptors. We reported previously that the RXR-type dibasic motif in the distal C-terminal tail of an HIV coreceptor GPR15 negatively regulates the cell surface expression by mediating the coatomer protein I complex-dependent retrograde transport to the endoplasmic reticulum (ER). Here we demonstrate that another pair of basic residues (Arg³¹⁰-Arg³¹¹) in the membrane-proximal region of the C-terminal tail plays a pivotal role in mediating the anterograde trafficking of GPR15. The Ala mutation of the C-terminal membrane-proximal basic residues (MPBRs) (R310/311A) abolished the O-glycosylation and cell surface expression of GPR15. The subcellular fractionation and immunocytochemistry assays indicated that the R310/311A mutant was more localized in the ER but much less in the trans-Golgi when compared with the wild-type GPR15, suggesting the positive role of Arg³¹⁰-Arg³¹¹ in the ER-to-Golgi transport of GPR15. Sequence analysis on human GPCRs showed that the basic residues are frequent in the membrane-proximal region of the C-terminal tail. Similar to GPR15, mutation of the C-terminal MPBRs resulted in a marked reduction of the cell surface expression in multiple different GPCRs. Our results suggest that the C-terminal MPBRs are critically involved in mediating the anterograde trafficking of a broad range of membrane proteins, including GPCRs.     

Characterization of proline-containing α-helix (helix F model of bacteriorhodopsin) by molecular dynamics studies

Many of the bilayer spanning segments of membrane transport proteins contain proline residues, and most of them are believed to occur in α-helical form. A proline residue in the middle of an α-helix is known to produce a bend in the helix, and recent studies have focused on characterizing such a bend at atomic level. In the present case, molecular dynamics (MD) studies are carried out on helix F model of bacteriorhodopsin (BR) Ace-(Ala)₇-Trp-(Ala)₂-Tyr-Pro-(Ala)₂-Trp-(Ala)₈-NHMe and compared with Ace-(Ala)₇-Trp-(Ala)₂-Tyr-(Ala)₃-Trp-(Ala)₈-NHMe in which the proline is replaced by alanine. The bend in the helix is characterized by structural parameters such as kink angle (α), wobble angle (θ), virtual torsion angle (ρ), and the hydrogen bond distance d (O_p?3 … N_p+1). The average values and the flexibility involved in these parameters are evaluated. The correlation among the bend related parameters are estimated. The equilibrium side chain orientations of tryptophan and tyrosine residues are discussed and compared with those found in the recently proposed model of bacteriorhodopsin. Finally, a detailed characterization of the bend in terms of secondary structures such as α_I, α_II and goniometric helices are discussed, which can be useful in the interpretation of the experimental results on the secondary structures of membrane proteins involving the proline residue. © 1993 Wiley-Liss, Inc.     

Effect of membrane tension on the electric field and dipole potential of lipid bilayer membrane

The dipole potential of lipid bilayer membrane controls the difference in permeability of the membrane to oppositely charged ions. We have combined molecular dynamics (MD) simulations and experimental studies to determine changes in electric field and electrostatic potential of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer in response to applied membrane tension. MD simulations based on CHARMM36 force field showed that electrostatic potential of DOPC bilayer decreases by ~45mV in the physiologically relevant range of membrane tension values (0 to 15dyn/cm). The electrostatic field exhibits a peak (~0.8×10(9)V/m) near the water/lipid interface which shifts by 0.9? towards the bilayer center at 15dyn/cm. Maximum membrane tension of 15dyn/cm caused 6.4% increase in area per lipid, 4.7% decrease in bilayer thickness and 1.4% increase in the volume of the bilayer. Dipole-potential sensitive fluorescent probes were used to detect membrane tension induced changes in DOPC vesicles exposed to osmotic stress. Experiments confirmed that dipole potential of DOPC bilayer decreases at higher membrane tensions. These results are suggestive of a potentially new mechanosensing mechanism by which mechanically induced structural changes in the lipid bilayer membrane could modulate the function of membrane proteins by altering electrostatic interactions and energetics of protein conformational states.     

Noninvasive cellular internalization of silver molecules by chitosan nanoneedles: a novel nanocarrier

We explore with molecular modeling, dynamics simulations, and a statistical model the ability of chitosan nanoneedles (CNNs) to be internalized into a model lipid bilayer as a function of their length, keeping in view of their applications in the field of biomedicine for advanced targeted drug delivery. In this study, we have computationally modeled and studied the structural geometry and the stability of CNNs formed by 4, 6, and 8 subunits. We reported the molecular surface analysis of the modeled CNNs along with molecular dynamic (MD) simulations studies toward revealing the noninvasive cellular internalization potential of these CNNs and a case study has been carried to study the ability of CNNs to translocate silver nanoparticles across membrane. The present results are strongly in support of further exploration of 8 subunits based CNNs for their application as target drug delivery vehicles. The hydrophilicity of the CNNs has been attributed as one of the key factors responsible for the internalization process. Moreover, our MD simulation studies marched the ability of CNNs to translocate silver nanoparticles through biological membrane in a similar manner that resembles cell-penetrating peptides.     

Lipid bilayer molecular dynamics study of lipid-derived agonists of the putative cannabinoid receptor, GPR55

Both L-α-lysophosphatidylinositol (LPI) and 2-arachidonoyl-sn-glycero-3-phosphoinositol (2-AGPI) have been reported to activate the putative cannabinoid receptor, GPR55. Recent microsecond time-scale molecular dynamics (MD) simulations and isothiocyanate covalent labeling studies have suggested that a transmembrane helix 6/7 (TMH6/7) lipid pathway for ligand entry may be necessary for interaction with cannabinoid receptors. Because LPI and 2-AGPI are lipid-derived ligands, conformations that each assumes in the lipid bilayer are therefore likely important for their interaction with GPR55. We report here the results of 70 ns NAMD molecular dynamics (MD) simulations of LPI and of 2-AGPI in a fully hydrated bilayer of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). These simulations are compared with a 70 ns simulation of the cannabinoid CB1 receptor endogenous ligand, N-arachidonoylethanolamine (anandamide, AEA) in a POPC bilayer. These simulations revealed that (1) LPI and 2-AGPI sit much higher in the bilayer than AEA, with inositol headgroups that can at times be solvated completely by water; (2) the behavior of the acyl chains of AEA and 2-AGPI are similar in their flexibilities in the bilayer, while the acyl chain of LPI has reduced flexibility; and (3) both 2-AGPI and LPI can adopt a tilted headgroup orientation by hydrogen bonding to the phospholipid phosphate/glycerol groups or via intramolecular hydrogen bonding. This tilted head group conformation (which represents over 40% of the conformer population of LPI (42.2 ± 3.3%) and 2-AGPI (43.7 ± 1.4%)) may provide a low enough profile in the lipid bilayer for LPI and 2-AGPI to enter GPR55 via the putative TMH6/7 entry port.     

Depth of α-synuclein in a bilayer determined by fluorescence, neutron reflectometry, and computation

α-Synuclein (α-syn) membrane interactions are implicated in the pathogenesis of Parkinson's disease. Fluorescence and neutron reflectometry (NR) measurements reveal that α-syn penetrates ～9–14 Å into the outer leaflet of the bilayer, with a substantial portion of the membrane-bound polypeptide extending into the aqueous solvent. For the first time, to our knowledge, we used NR to obtain direct quantitative evidence of α-syn-induced membrane thinning. To examine the effect of specific residues on membrane penetration depths, we used a series of W4-containing N-terminal peptides. We identified that the first 15 residues (P15) nearly recapitulate the features of the full-length protein (i.e., partition constants, molecular mobility, and insertion of the W4 side chain into the bilayer), and found that as few as the first four N-terminal residues are sufficient for vesicle binding. Although at least one imperfect amphipathic repeat sequence (KAKEGV) is required for α-helical formation, secondary structural formation has little effect on membrane affinity. To develop an N-terminal α-syn model for bilayer interactions, we performed molecular-dynamics simulations of the P15 peptide submerged in a bilayer. The simulation results are highly consistent with experimental data indicating a broad low-energy region (8.5–14.5 Å) for W4 insertion.