G protein-coupled receptor 21 in macrophages: An in vitro study.

GPR21 is an orphan and constitutively active receptor belonging to the superfamily of G-Protein Coupled Receptors (GPCRs). GPR21 couples to the Gq family of G proteins and is expressed in macrophages. Studies of GPR21 knock-out mice indicated that GPR21 may be involved in promoting macrophage migration. The aim of this study was to evaluate the role of GPR21 in human macrophages, analyzing (i) its involvement in cell migration and cytokine release and (ii) the consequence of its pharmacological inhibition by using the inverse agonist GRA2. THP-1 cells were activated and differentiated into either M1 or M2 macrophages. GPR21 expression was evaluated at gene and protein level, the signalling pathway was investigated by an IP1 assay, and cytokine release by ELISA. Cell migration was detected by the Boyden chamber migration assay, performed on macrophages derived from both the THP-1 cell line and human peripheral blood monocytes. In addition, we compared the effect of the pharmacological inhibition of GPR21 with the effect of the treatment with a specific GPR21 siRNA to downregulate the receptor expression, thus confirming that GRA2 acts as an inverse agonist of GPR21. GRA2 does not affect cell viability at the tested concentrations, but significantly reduces the release of TNF-α and IL-1ß from M1 macrophages. The analysis of the migratory ability highlighted opposite effects of GRA2 on M1 and M2 macrophages since it decreased M1, while it promoted M2 cell migration. Therefore, the pharmacological inhibition of GPR21 could be of interest for pathological conditions characterized by low grade chronic inflammation.

GPR21 Inhibition Increases Glucose-Uptake in HepG2 Cells.

GPR21 is a constitutively active, orphan, G-protein-coupled receptor, with in vivo studies suggesting its involvement in the modulation of insulin sensitivity. However, its precise contribution is not fully understood. As the liver is both a major target of insulin signalling and critically involved in glucose metabolism, the aim of this study was to examine the role of GPR21 in the regulation of glucose uptake and production in human hepatocytes. In particular, HepG2 cells, which express GPR21, were adopted as cellular models. Compared with untreated cells, a significant increase in glucose uptake was measured in cells treated with siRNA to downregulate GPR21 expression or with the GPR21-inverse agonist, GRA2. Consistently, a significantly higher membrane translocation of GLUT-2 was measured under these conditions. These effects were accompanied by an increased ratio of phAKT(Ser473)/tot-AKT and phGSK-3ß(Ser9)/tot-GSK-3ß, thus indicating a marked activation of the insulin signalling pathway. Moreover, a significant reduction in ERK activation was observed with GPR21 inhibition. Collectively, these results indicate that GPR21 mediates the negative effects on glucose uptake by the liver cells. In addition, they suggest that the pharmacological inhibition of GPR21 could be a novel strategy to improve glucose homeostasis and counteract hepatic insulin resistance.

The signalling mechanisms of a novel mitochondrial complex I inhibitor prevent lipid accumulation and attenuate TNF-α-induced insulin resistance in vitro.

RTC-1 has recently been identified as a member of a new class of anti-diabetic compounds acting through the inhibition of complex I of the mitochondrial respiratory chain (NADH:ubiquinone oxidoreductase) to improve glucose handling and inhibit weight gain in mice fed a high-fat diet (HFD). The exact mechanism by which the reduced activity of NADH:ubiquinone oxidoreductase, in response to RTC-1, promotes these improved metabolic parameters remains to be established. Through extensive in vitro analysis, new molecular insights into these downstream signalling pathways have been obtained. RTC-1-induced inhibition of NADH:ubiquinone oxidoreductase was found to promote glucose uptake in C2C12 myotubes in vitro, through the activation of the Akt substrate of 160kDa (AS160), in response to the increased activity of Akt and AMP-activated protein kinase (AMPK). RTC-1-induced phosphorylation of the AMPK substrate, acetyl-CoA carboxylase (ACC) in vitro, was associated with a decrease in lipid accumulation in 3T3-L1 adipocytes and murine mesenchymal stromal cells (MSC). The novel compound also prevented tumour necrosis factor-alpha (TNF-α)-induced insulin resistance and demonstrated insulin sensitising effects in C2C12 myotubes. Taken together, these results present a systematic analysis of the signalling mechanisms responsible for the potent anti-diabetic and anti-obesogenic effects of this modulator of mitochondrial function, strengthening the potential use of such compounds for the treatment of type 2 diabetes mellitus (T2DM).

Regulating the effects of GPR21, a novel target for type 2 diabetes.

Type 2 diabetes is a chronic metabolic disorder primarily caused by insulin resistance to which obesity is a major contributor. Expression levels of an orphan G protein-coupled receptor (GPCR), GPR21, demonstrated a trend towards a significant increase in the epididymal fat pads of wild type high fat high sugar (HFHS)-fed mice. To gain further insight into the potential role this novel target may play in the development of obesity-associated type 2 diabetes, the signalling capabilities of the receptor were investigated. Overexpression studies in HEK293T cells revealed GPR21 to be a constitutively active receptor, which couples to Gαq type G proteins leading to the activation of mitogen activated protein kinases (MAPKs). Overexpression of GPR21 in vitro also markedly attenuated insulin signalling. Interestingly, the effect of GPR21 on the MAPKs and insulin signalling was reduced in the presence of serum, inferring the possibility of a native inhibitory ligand. Homology modelling and ligand docking studies led to the identification of a novel compound that inhibited GPR21 activity. Its effects offer potential as an anti-diabetic pharmacological strategy as it was found to counteract the influence of GPR21 on the insulin signalling pathway.

Novel mitochondrial complex I inhibitors restore glucose-handling abilities of high-fat fed mice.

Metformin is the main drug of choice for treating type 2 diabetes, yet the therapeutic regimens and side effects of the compound are all undesirable and can lead to reduced compliance. The aim of this study was to elucidate the mechanism of action of two novel compounds which improved glucose handling and weight gain in mice on a high-fat diet. Wildtype C57Bl/6 male mice were fed on a high-fat diet and treated with novel, anti-diabetic compounds. Both compounds restored the glucose handling ability of these mice. At a cellular level, these compounds achieve this by inhibiting complex I activity in mitochondria, leading to AMP-activated protein kinase activation and subsequent increased glucose uptake by the cells, as measured in the mouse C2C12 muscle cell line. Based on the inhibition of NADH dehydrogenase (IC50 27µmolL(-1)), one of these compounds is close to a thousand fold more potent than metformin. There are no indications of off target effects. The compounds have the potential to have a greater anti-diabetic effect at a lower dose than metformin and may represent a new anti-diabetic compound class. The mechanism of action appears not to be as an insulin sensitizer but rather as an insulin substitute.

Analysis of the effect of a novel therapeutic for type 2 diabetes on the proteome of a muscle cell line.

Elevated serum retinol-binding protein (RBP) concentration has been implicated in the development of insulin resistance and type 2 diabetes. Two series of small molecules have been designed to lower serum levels by reducing secretion of the transthyretin-RBP complex from the liver and enhancing RBP clearance through the kidney. These small molecules were seen to improve glucose and insulin tolerance tests and to reduce body weight gain in mice rendered diabetic through a high fat diet. A proteomics study was conducted to better understand the effects of these compounds in muscle cells, muscle being the primary site for energy expenditure. One lead compound, RTC-15, is seen to have a significant effect on proteins involved in fat and glucose metabolism. This could indicate that the compound is having a direct effect on muscle tissue to improve energy homeostasis as well as a whole body effect on circulating RBP levels. This newly characterized group of antidiabetic compounds may prove useful in the treatment and prevention of insulin resistance and obesity.

The effect of retinol binding protein on the proteome of C2C12 muscle cells.

BACKGROUND: Retinol binding protein (RBP) and its membrane receptor, STRA6, are vital for the management of vitamin A in the body. Recently, elevated serum RBP levels have been implicated as a contributing factor to the development of insulin resistance and type 2 diabetes. However, conflicting opinions exist as to how these increased levels can cause insulin resistance. METHODS: In order to better understand the influences of RBP, a proteomic study was devised to determine the direct effect of RBP on a mouse muscle cell line, because the muscle is the principal site of insulin induced glucose uptake. C2C12 cells were treated with RBP for 16 h and the proteome analysed for alterations in protein abundance and phosphorylation by 2-DE. RESULTS: A number of changes were observed in response to retinol binding protein treatment, of which the most interesting were decreased levels of the phosphatase, protein phosphatase 1 ß. This phosphatase is responsible for regulating glycogen synthase and glycogen phosphorylase, the rate-limiting enzymes involved in glycogen storage and utilization. Retinol binding protein treatment resulted in increased phosphorylation and inhibition of glycogen synthase, with detrimental effects on insulin stimulated glycogen production in these cells. CONCLUSION: The results indicate that RBP may have a negative effect on energy storage in the cell and could contribute to the development of insulin resistance in muscle tissue. Understanding how retinol binding protein influences insulin resistance may reveal novel strategies to target this disease.

Production of functional human vitamin A transporter/RBP receptor (STRA6) for structure determination.

STRA6 is a plasma membrane protein that mediates the transport of vitamin A, or retinol, from plasma retinol binding protein (RBP) into the cell. Mutations in human STRA6 are associated with Matthew-Wood syndrome, which is characterized by severe developmental defects. Despite the obvious importance of this protein to human health, little is known about its structure and mechanism of action. To overcome the difficulties frequently encountered with the production of membrane proteins for structural determination, STRA6 has been expressed in Pichia pastoris as a fusion to green fluorescent protein (GFP), a strategy which has been a critical first step in solving the crystal structures of several membrane proteins. STRA6-GFP was correctly targeted to the cell surface where it bound RBP. Here we report the large-scale expression, purification and characterisation of STRA6-GFP. One litre of culture, corresponding to 175 g cells, yielded about 1.5 mg of pure protein. The interaction between purified STRA6 and its ligand RBP was studied by surface plasmon resonance-based binding analysis. The interaction between STRA6 and RBP was not retinol-dependent and the binding data were consistent with a transient interaction of 1 mole RBP/mole STRA6.

Expression, characterization and ligand specificity of lipocalin-1 interacting membrane receptor (LIMR).

Human lipocalin-1 interacting membrane receptor (LIMR) was the first lipocalin receptor to be identified, as a specific receptor for lipocalin-1 (Lcn1). Subsequently LIMR has been reported to interact with other ligands as well, notably with the bovine lipocalin ß-lactoglobulin (BLG) and with the unrelated secretoglobin uteroglobin (UG). To study the ligand-binding behaviour of this prototypic lipocalin receptor in more detail, a system was developed for the recombinant expression of LIMR in Drosophila Schneider 2 (S2) cells, and for the subsequent solubilization and purification of the protein. The receptor forms dimers or larger oligomers when solubilized in n-dodecyl ß-D-maltoside (DDM). The full-length, functional receptor was captured onto a surface plasmon resonance (SPR) chip via an α-V5 antibody, and the binding of various potential ligands was followed in time. In this way, LIMR was shown to be highly specific for Lcn1, binding the lipocalin with low micromolar to high nanomolar affinity. No interactions with any of the other putative ligands could be detected, raising doubts about the physiological relevance of the reported binding of BLG and UG to the receptor.

The melanocortin 4 receptor: oligomer formation, interaction sites and functional significance.

This study involves the structural and functional properties of the recombinant melanocortin 4 receptor (MC(4)R) expressed in the HEK-293 cell line. Using co-immuno-purification approaches, the receptor appears to be an oligomer, which can be crosslinked through disulphide bonds involving a native cysteine residue (84) to give a dimeric species. This position is located near the cytosolic region of transmembrane segment 2 and it is suggested that this is an interacting interface between MC(4)R monomers. Using co-expression of the native protein and a C84A mutant, it appears that the receptor also forms higher order oligomers via alternative interfaces. Interestingly, disulphide crosslink formation does not occur if the receptor is uncoupled from its G-protein, even though the oligomeric state is preserved. This suggests that the conformational changes, which occur on activation, affect the TM2 interface. The pharmacology of the agonist, NDP-MSH, indicates that the MC(4)R retains high affinity for the ligand in the absence of the G-protein but occupancy for the ligand is increased. The data can be interpreted to suggest that the G-protein exerts a negative allosteric effect on the receptor. Co-expression of one receptor lacking the ability to signal with another, which cannot bind the agonist, restored ligand-dependent activation of the G-protein to situations in which neither receptor on its own could activate the G-protein. Such transactivation suggests meaningful cross talk between the receptor subunits in the oligomeric complex. These studies demonstrate further unique features of the MC(4)R.

Pharmacological chaperones increase the cell-surface expression of intracellularly retained mutants of the melanocortin 4 receptor with unique rescuing efficacy profiles.

Mutated versions of membrane proteins often fail to express at the plasma membrane, but instead are trapped in the secretory pathway, resulting in disease. The retention of these mutant proteins is thought to result from local misfolding, which prevents export from the ER (endoplasmic reticulum), targeting the receptor for degradation via the ER-associated quality control system. The rhodopsin-like G-protein-coupled MC4R (melanocortin 4 receptor) is an example of such a membrane protein. Over 100 natural MC4R mutations are linked with an obese phenotype and to date represent the most common monogenic cause of severe early-onset obesity. More than 80% of these mutations result in a substantial proportion of MC4R being retained intracellularly. If these receptors were expressed at the plasma membrane, many could be functional, as mutations often occur in regions distinct from those associated with ligand or G-protein binding. Our aim is to show proof of concept that selective compounds can rescue the function of MC4R mutants by increasing their cell-surface expression, and further to this, examine whether the rescue profile differs between mutants. Whole-cell ELISA and 96-well fluorescence-based assays with N-terminally HA (haemagglutinin)-tagged and C-terminally mCherry-tagged mutant MC4Rs were used to screen a number of novel MC4R-selective compounds. A total of four related compounds increased the cell-surface expression of wild-type and three intracellularly retained mutant MC4Rs, thus acting as pharmacological chaperones. There appears to be a unique rescue efficacy profile for each compound that does not correlate with potency, suggesting distinct receptor conformations induced by the different mutations. A degree of functionality of V50M and S58C was also rescued following relocation to the cell surface.

Fenretinide derivatives act as disrupters of interactions of serum retinol binding protein (sRBP) with transthyretin and the sRBP receptor.

Serum retinol binding protein (sRBP) is released from the liver as a complex with transthyretin (TTR), a process under the control of dietary retinol. Elevated levels of sRBP may be involved in inhibiting cellular responses to insulin and in generating first insulin resistance and then type 2 diabetes, offering a new target for therapeutic attack for these conditions. A series of retinoid analogues were synthesized and examined for their binding to sRBP and their ability to disrupt the sRBP-TTR and sRBP-sRBP receptor interactions. A number inhibit the sRBP-TTR and sRBP-sRBP receptor interactions as well as or better than Fenretinide (FEN), presenting a potential novel dual mechanism of action and perhaps offering a new therapeutic intervention against type 2 diabetes and its development. Shortening the chain length of the FEN derivative substantially abolished binding to sRBP, indicating that the strength of the interaction lies in the polyene chain region. Differences in potency against the sRBP-TTR and sRBP-sRBP receptor interactions suggest variant effects of the compounds on the two loops of sRBP guarding the entrance of the binding pocket that are responsible for these two protein-protein interactions.

An analysis of the phosphoproteome of immune cell lines exposed to the immunomodulatory mycotoxin deoxynivalenol.

The mycotoxin deoxynivalenol (DON) commonly contaminates cereal grains. It is ubiquitous in the Western European diet, although chronic, low-dose effects in humans are not well described, but immunotoxicity has been reported. In this study, two-dimensional gel electrophoresis was used to identify phosphoproteomic changes in human B (RPMI1788) and T (Jurkat E6.1) lymphocyte cell lines after exposure to modest concentrations of DON (up to 500ng/mL) for 24h. Proteins identified as having altered phosphorylation state post-treatment (C-1-tetrahydrofolate synthase, eukaryotic elongation factor 2, nucleoside diphosphate kinase A, heat shock cognate 71kDa protein, eukaryotic translation initiation factor 3 subunit I and growth factor receptor-bound protein 2) are involved in regulation of metabolic pathways, protein biosynthesis and signaling transduction. All exhibited a greater than 1.4-fold change, reproducible in three separate experiments consisting of 36 gels in total. Flow cytometry validated the observations for eukaryotic elongation factor 2 and growth factor receptor-bound protein 2. These findings provide further insights as to how low dose exposure to DON may affect human immune function and may have potential as mechanism-based phosphoprotein biomarkers for DON exposure.

Proteomic analysis of the effects of the immunomodulatory mycotoxin deoxynivalenol.

The mycotoxin deoxynivalenol (DON) contaminates cereals worldwide and is a common contaminant in the Western European diet. At high doses, DON induces acute gastrointestinal toxicity; chronic, low-dose effects in humans are not well described, but immunotoxicity has been reported. In this study, 2-DE was used to identify proteomic changes in human B (RPMI1788) and T (JurkatE6.1) lymphocyte cell lines after exposure to minimally toxic concentrations (up to 500 ng/mL) for 24 h. Proteins which changed their abundance post treatment, by a greater than 1.4-fold change reproducible in three separate experiments consisting of 36 gels in total, are ubiquitin carboxyl-terminal hydrolase isozyme L3, proteasome subunit ß type-4 and α type-6, inosine-5'-monophosphate dehydrogenase 2, GMP synthase, microtubule-associated protein RP/EB family member 1 (EB1), RNA polymerases I, II, III subunit ABC1, triosephosphate isomerase and transketolase. Flow cytometry was used to validate changes to protein expression, except for EB1. These findings provide insights as to how low-dose exposure to DON may affect human immune function and may provide mechanism-based biomarkers for DON exposure.

Interactions of the melanocortin-4 receptor with the peptide agonist NDP-MSH.

Melanocortin-4 receptor (MC4R) has an important regulatory role in energy homeostasis and food intake. Peptide agonists of the MC4R are characterized by the conserved sequence His(6)-Phe(7)-Arg(8)-Trp(9), which is crucial for their interaction with the receptor. This investigation utilized the covalent attachment approach to identify receptor residues in close proximity to the bound ligand [Nle(4),D-Phe(7)]melanocyte-stimulating hormone (NDP-MSH), thereby differentiating between residues directly involved in ligand binding and those mutations that compromise ligand binding by inducing conformational changes in the receptor. Also, recent X-ray structures of G-protein-coupled receptors were utilized to refine a model of human MC4R in the active state (R(*)), which was used to generate a better understanding of the binding mode of the ligand NDP-MSH at the atomic level. The mutation of residues in the human MC4R--such as Leu106 of extracellular loop 1, and Asp122, Ile125, and Asp126 of transmembrane (TM) helix 3, His264 (TM6), and Met292 (TM7)--to Cys residues produced definitive indications of proximity to the side chains of residues in the core region of the peptide ligand. Of particular interest was the contact between D-Phe(7) on the ligand and Ile125 of TM3 on the MC4R. Additionally, Met292 (TM7) equivalent to Lys(7.45) (Ballesteros numbering scheme) involved in covalently attaching retinal in rhodopsin is shown to be in close proximity to Trp(9). For the first time, the interactions between the terminal regions of NDP-MSH and the receptor are described. The amino-terminus appears to be adjacent to a series of hydrophilic residues with novel interactions at Cys196 (TM5) and Asp189 (extracellular loop 2). These interactions are reminiscent of sequential ligand binding exhibited by the beta(2)-adrenergic receptor, with the former interaction being equivalent to the known interaction involving Ser204 of the beta(2)-adrenergic receptor.

A site-directed cross-linking approach to the characterization of subunit E-subunit G contacts in the vacuolar H+-ATPase stator.

To operate as a rotary motor, the ATP-hydrolyzing domain of the vacuolar H(+)-ATPase must be connected to a fixed structure in its membrane-bound proton pump domain by a mechanical stator. Although low-resolution structural data and spectroscopic analysis indicate that a filament-like subunit E/subunit G heterodimer performs this role, more detailed information about the relative arrangement of these subunits is limited. We have used a site-directed cross-linking approach to show that, in both bacterial and yeast V-type ATPases, the N-terminal alpha-helical segments of the G and E subunits are closely aligned over a distance of up to 40 A. Furthermore, cross-linking coupled to mass spectrometry shows that the C-terminal end of G is anchored at the C-terminal globular domain of subunit E. These data are consistent with a stator model comprising two approximately 150 A long parallel alpha-helices linked to each other at both ends, stabilized by a coiled-coil arrangement and capped by the globular C-terminal domain of E that connects the cytoplasmic end of the helical structure to the V-ATPase catalytic domain.

Expression and characterization of recombinant human retinol-binding protein in Pichia pastoris.

Plasma retinol-binding protein (RBP4) is the principal carrier of vitamin A in blood. Recent studies have suggested that RBP4 may have also a role in insulin resistance. To date the recombinant protein is usually produced by refolding inclusion bodies in Escherichia coli. Here we report the expression and characterization of recombinant human plasma RBP4 using the Pichia pastoris expression system. Simple and rapid purification allowed us to obtain 5mg/L of purified protein from the fermentation supernatant with no need to perform denaturing and refolding steps. The identity of the protein was verified by ion-trap MS and Western blotting. The functionality of recombinant RBP4, i.e., the binding to its physiologic ligand, retinol, and the interaction with transthyretin (TTR), was tested by fluorimetric and pull-down assays, respectively. The apparent dissociation constant for retinol to the recombinant protein of 2 x 10(-7)M was consistent with published data for native human protein. The recombinant protein interacted specifically with TTR. These results suggest that expression of recombinant human RBP4 in P. pastoris provides an efficient source of fully functional protein in soluble form for biochemical and biophysical studies.

Expression and structural characterization of peripherin/RDS, a membrane protein implicated in photoreceptor outer segment morphology.

Peripherin/RDS is a member of the tetraspanin family of integral membrane proteins and plays a major role in the morphology of photoreceptor outer segments. Peripherin/RDS has a long extracellular loop (hereafter referred to as the LEL domain), which is vital for its function. Point mutations in the LEL domain often lead to impaired photoreceptor formation and function, making peripherin/RDS an important drug target. Being a eukaryotic membrane protein, acquiring sufficient peripherin/RDS for biophysical characterisation represents a significant challenge. Here, we describe the expression and characterisation of peripherin/RDS in Drosophila melangolaster Schneider (S2) insect cells and in the methylotrophic yeast Pichia pastoris. The wild-type peripherin/RDS and the retinitis pigmentosa causing P216L mutant from S2 cells are characterised using circular dichroism (CD) spectroscopy. The structure of peripherin/RDS and of a pathogenic mutant is assessed spectroscopically for the first time. These findings are evaluated in relation to a three-dimensional model of the functionally important LEL domain obtained by protein threading.

Proteomic analysis of IgE-mediated secretion by LAD2 mast cells.

The LAD2 cell line is a relatively recent addition to the range of mast cell analogues and is of particular importance as it is the only human analogue which can be stimulated to degranulate in an IgE-dependent manner. Mast cells are tissue-based effector cells which have historically been shown to play an important role in the adaptive immune response, though there is now gathering evidence of their significance as a component of the innate immune system. These functions can be attributed to the ability of mast cells to regulate secretion of a wide variety of potent biologically active mediators through immediate and delayed responses. This well-orchestrated secretory mechanism of the mast cell makes it an ideal model in which to study this event. In this investigation, two-dimensional electrophoresis was employed as part of the proteomic characterization of the LAD2 human mast cell line, focusing in particular on a global analysis of membrane protein relocation after an IgE-mediated stimulatory event. This investigation has identified six membrane-associated protein spots which became phosphorylated upon IgE-mediated activation, 31 protein spots which displayed consistent recruitment to the membrane fraction, and three which were consistently lost from the soluble fraction. The scenario which emerges reveals a series of substantial changes which affect every compartment of the cell, providing evidence for a coordinated response to a secretory stimulus.

Electrophoretic field gradient focusing with on-column detection by fluorescence quenching.

Native, uncoloured, proteins can be focused in a column containing a fluorescent packing material, using hydrodynamic flow and a counteracting non-linear electric field, and imaged along the length of the channel by fluorescence quenching.