Homo- and hetero-oligomerization of G-protein-coupled receptors (GPCRs) were examined in HEK-293 cells using two variants of bioluminescence resonance energy transfer (BRET). BRET(2) (a variant of BRET) offers greatly improved separation of the emission spectra of the donor and acceptor moieties compared with traditional BRET. Previously recorded homo-oligomerization of the human delta-opioid receptor was confirmed using BRET(2). Homo-oligomerization of the kappa-opioid receptor was observed using both BRET techniques. Both homo- and hetero-oligomers, containing both delta- and kappa-opioid receptors, were unaffected by the presence of receptor ligands. BRET detection of opioid receptor homo- and hetero-oligomers required expression of 50,000-100,000 copies of the receptor energy acceptor construct per cell. The effectiveness of delta-kappa-opioid receptor hetero-oligomer formation was as great as for homomeric interactions. The capacity of the two opioid receptors to form oligomeric complexes with the beta(2)-adrenoceptor was also assessed. Although such interactions were detected, at least 250,000 copies per cell of the energy acceptor were required. Requirement for high levels of receptor expression was equally pronounced in attempts to measure hetero-oligomer formation between the kappa-opioid receptor and the thyrotropin-releasing hormone receptor-1. These studies indicate that constitutively formed homo- and hetero-oligomers of opioid receptor subtypes can be detected in living cells containing less than 100,000 copies of the receptors. However, although hetero-oligomeric interactions between certain less closely related GPCRs can be detected, they appear to be of lower affinity than homo- or hetero-oligomers containing closely related sequences. Interactions recorded between certain GPCR family members in heterologous expression systems are likely to be artefacts of extreme levels of overexpression.

]enkephalin nor the inverse agonist ligand ICI174864 were able to modulate the oligomerization status of this receptor. Interactions between co-expressed ␦-opioid receptors and ␤ 2 -adrenoreceptors were observed in co-immunoprecipitation studies. Such hetero-oligomers could also be detected using bioluminescence resonance energy transfer although the signal obtained was substantially smaller than for homo-oligomers of either receptor type. Signal corresponding to the ␦-opioid receptor-␤ 2 -adrenoreceptor hetero-oligomer was increased in the presence of agonist for either receptor. However, substantial levels of this hetero-oligomer were not detected at the cell surface using time-resolved fluorescence resonance energy transfer. These studies demonstrate that, following transient transfection of HEK293 cells, constitutively formed oligomers of the human ␦-opioid receptor can be detected by a variety of approaches. However, these are not regulated by ligand occupancy. They also indicate that time-resolved fluorescence resonance energy transfer represents a means to detect such oligomers at the cell surface in populations of intact cells.Recent studies have started to provide a significant body of evidence to support a concept of constitutive homo-oligomerization of a range of G protein-coupled receptors (GPCRs) 1 (1, 2).GPCRs for which such evidence exists include the ␤ 2 -adrenoreceptor (3, 4), the D 2 dopamine receptor (5), the M 3 muscarinic acetylcholine receptor (6), the V 2 vasopressin receptor (7), the ␦-opioid (8, 9) and -opioid receptors (9), the histamine H 2 receptor (10), and the CCR5 receptor (11). Furthermore, recent evidence has also indicated a requirement for the constitutive hetero-oligomerization of distinct GPCRs, such as between the GABA B R1 and GABA B R2 receptors, to generate a functional receptor expressed at the cell surface (12). Two important issues, however, remain contentious. The first of these is whether ligand occupancy alters the extent of GPCR oligomerization, and the second is the likely extent of GPCR hetero-oligomerization. In a range of reports, GPCR homo-oligomerization has been reported to be increased (3, 4, 11), decreased (8), or unaffected (6, 7) by the addition of receptor ligands. Similarly, in GPCR heterodimerization studies, interactions have been indicated to be unaffected (12), regulated (13), or almost entirely dependent upon (14) the addition of receptor agonists and hetero-oligomers have recently been reported to form between quite distinct (14), as well as between closely related (12, 13), GPCR sequences. The earliest studies on GPCR oligomerization relied on the capacity to co-immunoprecipitate co-expressed but differentially epitope-tagged forms of a GPCR (see Ref. 15 for review). Because of the hydrophobic nature of the seven trans-plasma membrane helices of GPCR family members, care must be taken, however, to exclude nonspecific interactions between GPCR pairs resulting from detergent dissolution of cellular membranes. More recent studies have employed vari...

The interactions of isoleucyl-tRNA synthetase (IleRS,E

The therapeutic potential of urease inhibition of Helicobacter pylori has been studied by examining the effect of the potent urease inhibitor, fluorofamide (N-(diaminophosphinyl)-4-fluorobenzenamide), on urease activity and bacterial survival in vivo and in vitro. In culture, acid protection in H. pylori was shown to be due to changes in the pH of the medium brought about by the release of ammonia. Both the acid protection and the ammonia release were completely blocked by fluorofamide at low doses (ED50 = approximately 100 nM). However, fluorofamide was unstable under acidic conditions (T1/2 = 5.7 min at pH 2). Despite this, fluorofamide was the best available compound to test in vivo. In ferrets naturally infected with H. mustelae, a single dose (50 mg/kg, per os) of fluorofamide completely inhibited bacterial urease. In repeat dosing studies, fluorofamide (50 mg/kg per os, three times a day) was compared with the Helicobacter triple therapy regime (amoxycillin, metronidazole, and bismuth subcitrate). Fluorofamide failed to eradicate the H. mustelae infection, compared to 80% eradication with triple therapy. However, histological samples showed a profound reduction in bacterial numbers following fluorofamide treatment. A combination of fluorofamide and amoxycillin was dosed to ferrets (seven days of treatment with 50 mg/kg fluorofamide plus 10 mg/kg amoxycillin per os twice a day); however, this failed to eradicate the infection, despite there being a reduction in bacterial numbers in 3/5 ferrets after 21 days after dosing stopped. It was concluded that urease inhibitors (either alone or in combination with antibiotics) are unlikely to have therapeutic potential for Helicobacter pylori infections. This is probably because, in vivo, some bacteria (perhaps dormant forms) are not entirely dependent upon urease for survival. However, given the acid instability of fluorofamide, the possibility that more stable urease inhibitors might have therapeutic potential, cannot be excluded.

No abstract

Pseudomonic acid A (PS-A)1 is a potent antibiotic that acts through inhibition of bacterial isoleucyl-tRNA synthetase (IleRS, E) (1, 2). As part of an effort to understand the precise mechanism of the interaction of PS-A and its analogues with IleRS we have undertaken a detailed characterization of the kinetics and thermodynamics of substrate and inhibitor binding by this enzyme (3, 4). These experiments showed that PS-A analogues are reversible, slow tight-binding inhibitors that form an highly stabilized inhibitor complex with overall dissociation constants in the picomolar range. Comparison of PS-A analogues with inhibitors that were non-hydrolyzable analogues of the normal activated enzyme intermdiate, Ile-AMP, showed that both classes of inhibitor bound to IleRS in a manner that was mutually exclusive with each other and with the substrates, L-isoleucine (Ile) and Mg⅐ATP, but that very different enzyme conformations were induced upon binding (as shown by differences in IleRS tryptophan fluorescence) (4). In this report, we describe experiments in which we attempted to gain additional information on the binding of substrates and inhibitors to IleRS using different approaches to the more classical techniques which we had previously employed (e.g. state-state and transient kinetics (3, 4)).One technique for investigating changes in the conformation of proteins as a result of ligand binding is to test their effect upon proteolysis (e.g. Ref. 5), and this has previously been shown to yield useful information on several aminoacyl-tRNA synthetases (e.g. Refs. 6 -9). Here, we used this approach in two distinct ways. First, the effect of substrate and inhibitor binding on IleRS proteolysis patterns was investigated in an attempt to identify protein determinants for binding. Second, a homogeneous real-time assay capable of monitoring ligand protection from IleRS proteolysis using fluorescence polarization was developed which could be potentially used to screen for new inhibitors.

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BackgroundLow levels of adiponectin, an adipocytokine with anti-diabetic, antiatherogenic and cardioprotective properties, is associated with increased risk of coronary disease in young men. Previous studies have demonstrated that smokeless tobacco is linked with a reduction of plasma adiponectin levels. However, the influence of smokeless tobacco (dipping tobacco) on plasma adiponectin levels still remains unknown. This study was conducted to assess the plasma adiponectin levels in young men who were using dipping tobacco.MethodsThis was a community based study, which consisted of 186 young lean healthy males aged 20 to 35 years. Among these, 96 men were dipping tobacco users (BMI = 23.07 ± 2.68) and 90 were non-dipping tobacco users (BMI = 23.67 ± 1.46). Serum adiponectin levels were assessed by Enzyme Linked ImmunoSorbent Assay (ELISA).ResultsA statistically significant difference in the mean adiponectin level between tobacco dipper and non-dipper groups was observed (p = 0.0001). A significant difference between the two groups was also observed in baseline parameters including triglyceride and random blood sugar levels (p < 0.05). However, no significant difference was observed between the two groups in other clinical parameters.ConclusionsFindings of this study suggest that dipping tobacco use was significantly associated with low level of adiponetin in community dwelling young males. This emphasizes the importance of developing community intervention to reduce the use of dipping tobacco, which will reduce the tobacco associated disease burden in the community and will improve public health.