The ghrelin receptor isoforms (GHS-R1a and GHS-R1b) and GPR39 : an investigation into receptor dimerisation

Prostate cancer is the second most common cause of cancer-related deaths in Western males. Current diagnostic, prognostic and treatment approaches are not ideal and advanced metastatic prostate cancer is incurable. There is an urgent need for improved adjunctive therapies and markers for this disease. GPCRs are likely to play a significant role in the initiation and progression of prostate cancer. Over the last decade, it has emerged that G protein coupled receptors (GPCRs) are likely to function as homodimers and heterodimers. Heterodimerisation between GPCRs can result in the formation of novel pharmacological receptors with altered functional outcomes, and a number of GPCR heterodimers have been implicated in the pathogenesis of human disease. Importantly, novel GPCR heterodimers represent potential new targets for the development of more specific therapeutic drugs. Ghrelin is a 28 amino acid peptide hormone which has a unique n-octanoic acid post-translational modification. Ghrelin has a number of important physiological roles, including roles in appetite regulation and the stimulation of growth hormone release. The ghrelin receptor is the growth hormone secretagogue receptor type 1a, GHS-R1a, a seven transmembrane domain GPCR, and GHS-R1b is a C-terminally truncated isoform of the ghrelin receptor, consisting of five transmembrane domains. Growing evidence suggests that ghrelin and the ghrelin receptor isoforms, GHS-R1a and GHS-R1b, may have a role in the progression of a number of cancers, including prostate cancer. Previous studies by our research group have shown that the truncated ghrelin receptor isoform, GHS-R1b, is not expressed in normal prostate, however, it is expressed in prostate cancer. The altered expression of this truncated isoform may reflect a difference between a normal and cancerous state. A number of mutant GPCRs have been shown to regulate the function of their corresponding wild-type receptors. Therefore, we investigated the potential role of interactions between GHS-R1a and GHS-R1b, which are co-expressed in prostate cancer and aimed to investigate the function of this potentially new pharmacological receptor. In 2005, obestatin, a 23 amino acid C-terminally amidated peptide derived from preproghrelin was identified and was described as opposing the stimulating effects of ghrelin on appetite and food intake. GPR39, an orphan GPCR which is closely related to the ghrelin receptor, was identified as the endogenous receptor for obestatin. Recently, however, the ability of obestatin to oppose the effects of ghrelin on appetite and food intake has been questioned, and furthermore, it appears that GPR39 may in fact not be the obestatin receptor. The role of GPR39 in the prostate is of interest, however, as it is a zinc receptor. Zinc has a unique role in the biology of the prostate, where it is normally accumulated at high levels, and zinc accumulation is altered in the development of prostate malignancy. Ghrelin and zinc have important roles in prostate cancer and dimerisation of their receptors may have novel roles in malignant prostate cells. The aim of the current study, therefore, was to demonstrate the formation of GHS-R1a/GHS-R1b and GHS-R1a/GPR39 heterodimers and to investigate potential functions of these heterodimers in prostate cancer cell lines. To demonstrate dimerisation we first employed a classical co-immunoprecipitation technique. Using cells co-overexpressing FLAG- and Myc- tagged GHS-R1a, GHS-R1b and GPR39, we were able to co-immunoprecipitate these receptors. Significantly, however, the receptors formed high molecular weight aggregates. A number of questions have been raised over the propensity of GPCRs to aggregate during co-immunoprecipitation as a result of their hydrophobic nature and this may be misinterpreted as receptor dimerisation. As we observed significant receptor aggregation in this study, we used additional methods to confirm the specificity of these putative GPCR interactions. We used two different resonance energy transfer (RET) methods; bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET), to investigate interactions between the ghrelin receptor isoforms and GPR39. RET is the transfer of energy from a donor fluorophore to an acceptor fluorophore when they are in close proximity, and RET methods are, therefore, applicable to the observation of specific protein-protein interactions. Extensive studies using the second generation bioluminescence resonance energy transfer (BRET2) technology were performed, however, a number of technical limitations were observed. The substrate used during BRET2 studies, coelenterazine 400a, has a low quantum yield and rapid signal decay. This study highlighted the requirement for the expression of donor and acceptor tagged receptors at high levels so that a BRET ratio can be determined. After performing a number of BRET2 experimental controls, our BRET2 data did not fit the predicted results for a specific interaction between these receptors. The interactions that we observed may in fact represent ‘bystander BRET’ resulting from high levels of expression, forcing the donor and acceptor into close proximity. Our FRET studies employed two different FRET techniques, acceptor photobleaching FRET and sensitised emission FRET measured by flow cytometry. We were unable to observe any significant FRET, or FRET values that were likely to result from specific receptor dimerisation between GHS-R1a, GHS-R1b and GPR39. While we were unable to conclusively demonstrate direct dimerisation between GHS-R1a, GHS-R1b and GPR39 using several methods, our findings do not exclude the possibility that these receptors interact. We aimed to investigate if co-expression of combinations of these receptors had functional effects in prostate cancers cells. It has previously been demonstrated that ghrelin stimulates cell proliferation in prostate cancer cell lines, through ERK1/2 activation, and GPR39 can stimulate ERK1/2 signalling in response to zinc treatments. Additionally, both GHS-R1a and GPR39 display a high level of constitutive signalling and these constitutively active receptors can attenuate apoptosis when overexpressed individually in some cell types. We, therefore, investigated ERK1/2 and AKT signalling and cell survival in prostate cancer the potential modulation of these functions by dimerisation between GHS-R1a, GHS-R1b and GPR39. Expression of these receptors in the PC-3 prostate cancer cell line, either alone or in combination, did not alter constitutive ERK1/2 or AKT signalling, basal apoptosis or tunicamycin-stimulated apoptosis, compared to controls. In summary, the potential interactions between the ghrelin receptor isoforms, GHS-R1a and GHS-R1b, and the related zinc receptor, GPR39, and the potential for functional outcomes in prostate cancer were investigated using a number of independent methods. We did not definitively demonstrate the formation of these dimers using a number of state of the art methods to directly demonstrate receptor-receptor interactions. We investigated a number of potential functions of GPR39 and GHS-R1a in the prostate and did not observe altered function in response to co-expression of these receptors. The technical questions raised by this study highlight the requirement for the application of extensive controls when using current methods for the demonstration of GPCR dimerisation. Similar findings in this field reflect the current controversy surrounding the investigation of GPCR dimerisation. Although GHS-R1a/GHS-R1b or GHS-R1a/GPR39 heterodimerisation was not clearly demonstrated, this study provides a basis for future investigations of these receptors in prostate cancer. Additionally, the results presented in this study and growing evidence in the literature highlight the requirement for an extensive understanding of the experimental method and the performance of a range of controls to avoid the spurious interpretation of data gained from artificial expression systems. The future development of more robust techniques for investigating GPCR dimerisation is clearly required and will enable us to elucidate whether GHS-R1a, GHS-R1b and GPR39 form physiologically relevant dimers.

Aspects of tautomerism. Part III. A new dimerisation reaction of pseudo-acid chlorides

Pseudo-acid chlorides of five 4′-substituted o-benzoylbenzoic acids are converted into a mixture of dilactones with sodium iodide in acetone. The meso-isomer is always formed to a larger extent than the (±)-mixture. These results imply that the radicals involved are not planar.

GTP Cyclohydrolase I Gene Transfer Augments Intracellular Tetrahydrobiopterin in Human Endothelial Cells: Effects on Nitric Oxide Synthase Activity and Dimerisation.

Objectives: Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) activity. BH4 levels are regulated by de novo biosynthesis; the rate-limiting enzyme is GTP cyclohydrolase I (GTPCH). BH4 activates and promotes homodimerisation of purified eNOS protein, but the intracellular mechanisms underlying BH4-mediated eNOS regulation in endothelial cells remain less clear. We aimed to investigate the role of BH4 levels in intracellular eNOS regulation, by targeting the BH4 synthetic pathway as a novel strategy to modulate intracellular BH4 levels. Methods: We constructed a recombinant adenovirus, AdGCH, encoding human GTPCH. We infected human endothelial cells with AdGCH, investigated the changes in intracellular biopterin levels, and determined the effects on eNOS enzymatic activity, protein levels and dimerisation. Results: GTPCH gene transfer in EAhy926 endothelial cells increased BH4 >10-fold compared with controls (cells alone or control adenovirus infection), and greatly enhanced NO production in a dose-dependent, eNOS-specific manner. We found that eNOS was principally monomeric in control cells, whereas GTPCH gene transfer resulted in a striking increase in eNOS homodimerisation. Furthermore, the total amounts of both native eNOS protein and a recombinant eNOS–GFP fusion protein were significantly increased following GTPCH gene transfer. Conclusions: These findings suggest that GTPCH gene transfer is a valid approach to increase BH4 levels in human endothelial cells, and provide new evidence for the relative importance of different mechanisms underlying BH4-mediated eNOS regulation in intact human endothelial cells. Additionally, these observations suggest that GTPCH may be a rational target to augment endothelial BH4 and normalise eNOS activity in endothelial dysfunction states.

Dimerisation of cyclooctene using Grubbs' catalysts

The dimerisation of cyclooctene (COE) to 1,9-cyclohexadecadiene, a molecule of interest to the fragrance industry, has been achieved using ruthenium catalysts in organic solvents with significantly better selectivities (47-74%) and yields (39-60%) than previously reported (34% and 30%, respectively). Grubbs' first and second generation catalysts, the Hoveyda-Grubbs' catalyst and a phosphonium alkylidene catalyst were tested in a range of organic solvents and ionic liquids (ILs), including 1:1 IL/dichloromethane mixtures and biphasic IL + pentane systems. The best results (74% selectivity, 60% yield) were obtained using Grubbs' first generation catalyst in 1,2-dichloroethane. The formation of trimer, tetramer and other higher molecular mass products were found to be favoured at low catalyst loadings (0.77 mM). Studies of metathesis reactions using 1,9-cyclohexadecadiene as substrate indicated that the monomer-dimer and monomer-trimer reactions are faster than the dimer-dimer reaction. The use of IL media allowed for the recyclability of the catalyst, although a drop in the yield of dimer generally occurred after the first run. Heterogeneized catalysts, where the IL-catalyst system was immobilised onto silica, resulted in fast reactions leading to poor yields of dimer. (C) 2011 Elsevier B.V. All rights reserved.

Glycolaldehyde Formation via the Dimerisation of the Formyl Radical

Glycolaldehyde, the simplest monosaccharide sugar, has recently been detected in low- and high-mass star-forming cores. Following our previous investigation into glycolaldehyde formation, we now consider a further mechanism for the formation of glycolaldehyde that involves the dimerization of the formyl radical, HCO. Quantum mechanical investigation of the HCO dimerization process upon an ice surface is predicted to be barrierless and therefore fast. In an astrophysical context, we show that this mechanism can be very efficient in star-forming cores. It is limited by the availability of the formyl radical, but models suggest that only very small amounts of CO are required to be converted to HCO to meet the observational constraints.

Ribosome modulation factor protects Escherichia coli during heat stress, but this may not be dependent on ribosome dimerisation

The role of ribosome modulation factor (RMF) in protecting heat-stressed Escherichia coli cells was identified by the observation that cultures of a mutant strain lacking functional RMF (HMY15) were highly heat sensitive in stationary phase compared to those of the parent strain (W3110). No difference in heat sensitivity was observed between these strains in exponential phase, during which RMF is not synthesised. Studies by differential scanning calorimetry demonstrated that the ribosomes of stationary-phase cultures of the mutant strain had lower thermal stability than those of the parent strain in stationary phase, or exponential-phase ribosomes. More rapid breakdown of ribosomes in the mutant strain during heating was confirmed by rRNA analysis and sucrose density gradient centrifugation. Analyses of ribosome composition showed that the 100S dimers dissociated more rapidly during heating than 70S particles. While ribosome dimerisation is a consequence of the conformational changes caused by RMF binding, it may not therefore be essential for RMF-mediated ribosome stabilisation.

Organic reactions in ionic liquids: oxidative dimerisation of thioamides with phenyliodine(III) diacetate

The room temperature ionic liquid, 1-n-butylpyridinium tetrafluoroborate (BPyBF4), is used as a “green“ recyclable solvent for the oxidative dimerisation of thioamides with phenyliodine(III) diacetate which provides a facile, efficient and environmentally benign method for the synthesis of 3,5-diaryl-1,2,4-thiadiazoles.

Tethering for Selective Synthesis of 2,2′-Biphenols : the Acetal Method

2,2'-Biphenols are a large and diverse group of compounds with exceptional properties both as ligands and bioactive agents. Traditional methods for their synthesis by oxidative dimerisation are often problematic and lead to mixtures of ortho- and para-connected regioisomers. To compound these issues, an intermolecular dimerisation strategy is often inappropriate for the synthesis of heterodimers. The ‘acetal method’ provides a solution for these problems: stepwise tethering of two monomeric phenols enables heterodimer synthesis, enforces ortho regioselectivity and allows relatively facile and selective intramolecular reactions to take place. The resulting dibenzo[1,3]dioxepines have been analysed by quantum chemical calculations to obtain information about the activation barrier for ring flip between the enantiomers. Hydrolytic removal of the dioxepine acetal unit revealed the 2,2′-biphenol target.

Structure of a dimer of ageratochromene

A solid obtained in the dimerisation of ageratochromene (6,7-dimethoxy-2,2-dimethyl-1-benzopyran)(II) in the presence of acid has been shown to be 5,6,6a,6b,7,12b-hexahydro-1,2,10,11-tetramethoxy-5,5,7,7-tetramethylcyclopenta[1,2-c;5,4,3-de]bis[1]benzopyran (IV) by a study of its n.m.r. and mass spectra.

Three-dimensional hybrid networks based on aspartic acid

Three-dimensional achiral coordination polymers of the general formula M2(D, l-NHCH (COO)CH2COO)2·C4H4N2 where M = Ni and Co and pyrazine acts as the linker molecule have been prepared under hydrothermal conditions starting with [M(L-NHCH(COO)CH2COO)·3H2O] possessing a helical chain structure. A three-dimensional hybrid compound of the formula Pb2.5[N{CH(COO) CH2COO}22H2O] has also been prepared hydrothermally starting with aspartic acid and Pb(NO3)2. In this lead compound, where a secondary amine formed by the dimerisation of aspartic acid acts as the ligand, there is two-dimensional inorganic connectivity and one-dimensional organic connectivity.

Role of molecular reorientation in the vitrification of molecular liquids: A Monte Carlo study of the liquid and glassy states of 2,2-dimethylbutane

The liquid and the glassy phases of 2,2-dimethylbutane have been investigated by isothermal isobaric ensemble Monte Carlo simulation. Thermodynamic Properties and radial distribution functions for both the liquid and the glass have been obtained. The radial distribution functions have been classified into three types based on the accessibility of the group. It has been shown that the structure of the Iiquid and the glass can be understood in terms of the above classification of the radial distribution functions. Molecular reorientation plays an important role in the structural rearrangement accompanying glass formation. As much as 35% of the contribution to the increase in the intermolecular interaction energy on vitrification is due to the reorientation of the neighbouring pairs of molecules. The observed changes in the dimerisation energy and the bonding energy distribution function are consistent with the observed structural changes.

Extent of Structural Asymmetry in Homodimeric Proteins: Prevalence and Relevance

Most homodimeric proteins have symmetric structure. Although symmetry is known to confer structural and functional advantage, asymmetric organization is also observed. Using a non-redundant dataset of 223 high-resolution crystal structures of biologically relevant homodimers, we address questions on the prevalence and significance of asymmetry. We used two measures to quantify global and interface asymmetry, and assess the correlation of several molecular and structural parameters with asymmetry. We have identified rare cases (11/223) of biologically relevant homodimers with pronounced global asymmetry. Asymmetry serves as a means to bring about 2:1 binding between the homodimer and another molecule; it also enables cellular signalling arising from asymmetric macromolecular ligands such as DNA. Analysis of these cases reveals two possible mechanisms by which possible infinite array formation is prevented. In case of homodimers associating via non-topologically equivalent surfaces in their tertiary structures, ligand-dependent mechanisms are used. For stable dimers binding via large surfaces, ligand-dependent structural change regulates polymerisation/depolymerisation; for unstable dimers binding via smaller surfaces that are not evolutionarily well conserved, dimerisation occurs only in the presence of the ligand. In case of homodimers associating via interaction surfaces with parts of the surfaces topologically equivalent in the tertiary structures, steric hindrance serves as the preventive mechanism of infinite array. We also find that homodimers exhibiting grossly symmetric organization rarely exhibit either perfect local symmetry or high local asymmetry. Binding of small ligands at the interface does not cause any significant variation in interface asymmetry. However, identification of biologically relevant interface asymmetry in grossly symmetric homodimers is confounded by the presence of similar small magnitude changes caused due to artefacts of crystallisation. Our study provides new insights regarding accommodation of asymmetry in homodimers.

Model for triplet state engineering in organic light emitting diodes

Engineering the position of the lowest triplet state (T-1) relative to the first excited singlet state (S-1) is of great importance in improving the efficiencies of organic light emitting diodes and organic photovoltaic cells. We have carried out model exact calculations of substituted polyene chains to understand the factors that affect the energy gap between S-1 and T-1. The factors studied are backbone dimerisation, different donor-acceptor substitutions, and twisted geometry. The largest system studied is an 18 carbon polyene which spans a Hilbert space of about 991 x 10(6). We show that for reverse intersystem crossing process, the best system involves substituting all carbon sites on one half of the polyene with donors and the other half with acceptors. (C) 2014 AIP Publishing LLC.

Enhancing hydrogen storage capacity of pyridine-based metal organic framework

Using first principles calculations, we show that the storage capacity as well as desorption temperature of MOFs can be significantly enhanced by decorating pyridine (a common linker in MOFs) by metal atoms. The storage capacity of metal-pyridine complexes are found to be dependent on the type of decorating metal atom. Among the 3d transition metal atoms, Sc turns out to be the most efficient storing unto four H-2 molecules. Most importantly, Sc does not suffer dimerisation on the surface of pyridine, keeping the storage capacity of every metal atom intact. Based on these findings, we propose a metal-decorated pyridine-based MOFs, which has potential to meet the required H-2 storage capacity for vehicular usage. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Mycobacterium tuberculosis Cell Division Protein, FtsE, is an ATPase in Dimeric Form

FtsE is one of the earliest cell division proteins that assembles along with FtsX at the mid-cell site during cell division in Escherichia coli. Both these proteins are highly conserved across diverse bacterial genera and are predicted to constitute an ABC transporter type complex, in which FtsE is predicted to bind ATP and hydrolyse it, and FtsX is predicted to be an integral membrane protein. We had earlier reported that the MtFtsE of the human pathogen, Mycobacterium tuberculosis, binds ATP and interacts with MtFtsX on the cell membrane of M. tuberculosis and E. coli. In this study, we demonstrate that MtFtsE is an ATPase, the active form of which is a dimer, wherein the participating monomers are held together by non-covalent interactions, with the Cys84 of each monomer present at the dimer interface. Under oxidising environment, the dimer gets stabilised by the formation of Cys84-Cys84 disulphide bond. While the recombinant MtFtsE forms a dimer on the membrane of E. coli, the native MtFtsE seems to be in a different conformation in the M. tuberculosis membrane. Although disulphide bridges were not observed on the cytoplasmic side (reducing environment) of the membrane, the two participating monomers could be isolated as dimers held together by non-covalent interactions. Taken together, these findings show that MtFtsE is an ATPase in the non-covalent dimer form, with the Cys84 of each monomer present in the reduced form at the dimer interface, without participating in the dimerisation or the catalytic activity of the protein.