The peptide hormone ghrelin is the only known protein modified with an O-linked octanoyl side group, which occurs on its third serine residue. This modification is crucial for ghrelin's physiological effects including regulation of feeding, adiposity, and insulin secretion. Despite the crucial role for octanoylation in the physiology of ghrelin, the lipid transferase that mediates this novel modification has remained unknown. Here we report the identification and characterization of human GOAT, the ghrelin O-acyl transferase. GOAT is a conserved orphan membrane-bound O-acyl transferase (MBOAT) that specifically octanoylates serine-3 of the ghrelin peptide. Transcripts for both GOAT and ghrelin occur predominantly in stomach and pancreas. GOAT is conserved across vertebrates, and genetic disruption of the GOAT gene in mice leads to complete absence of acylated ghrelin in circulation. The occurrence of ghrelin and GOAT in stomach and pancreas tissues demonstrates the relevance of GOAT in the acylation of ghrelin and further implicates acylated ghrelin in pancreatic function.G hrelin is a 28-aa peptide hormone produced principally by stomach tissue with an unusual acyl modification on its critical serine-3 residue. Ghrelin is the endogenous ligand for the growth hormone secretagogue receptor 1a (GHSR1a), and its acyl modification is critical for the activation of the GHSR1a (1). In addition to stimulating growth hormone release from pituitary, ghrelin also promotes food intake, carbohydrate utilization, and adiposity (2-5). Accordingly, ghrelin levels are modulated by changes in nutritional status such as feeding and fasting or exposure to high-fat diets (5, 6). Importantly, ghrelin is the only peptide hormone of peripheral tissue origin that increases food intake (2).More recent studies have identified roles for acylated ghrelin in regulating insulin secretion and blood glucose. Acylated ghrelin occurs in pancreas tissues, and GHSR1a receptor blockade with specific antagonists or treatments with antiserum against acylated ghrelin enhance glucose-induced increases in insulin release whereas acylated ghrelin decreases insulin release (5, 7-10). These observations have further implicated acylated ghrelin in the regulation of metabolism.In stomach tissue and in circulation, acylated forms of ghrelin are modified via an ester linkage with n-octanoic acid and to a lesser extent with decanoyl and decenoyl fatty acids (1, 3). Importantly, the acyl modification in ghrelin is essential for function, with octanoyl and decanoyl fatty acids being optimal (11). Ghrelin is highly conserved in vertebrates, and the third serine residue, which is uniquely modified by the ester-linked acyl group, occurs in all mammal, avian, and fish species (3).The enzyme(s) responsible for acylation of ghrelin has remained unknown. Work by Takada et al. (12) described that porcupine, an enzyme with structural similarities to membrane-bound O-acyl transferases (MBOAT), is required for serine-209 acylation with palmitoleic acid and for transport of...

The first endocannabinoid, anandamide, was discovered in 1992. Since then, two other endocannabinoid agonists have been identified, 2-arachidonyl glycerol and, more recently, noladin ether. Here, we report the identification and pharmacological characterization of a novel endocannabinoid, virodhamine, with antagonist properties at the CB1 cannabinoid receptor. Virodhamine is arachidonic acid and ethanolamine joined by an ester linkage. Concentrations of virodhamine measured by liquid chromatography atmospheric pressure chemical ionization-tandem mass spectrometry in rat brain and human hippocampus were similar to anandamide. In peripheral tissues that express the CB2 cannabinoid receptor, virodhamine concentrations were 2-to 9-fold higher than anandamide. In contrast to previously described endocannabinoids, virodhamine was a partial agonist with in vivo antagonist activity at the CB1 receptor. However, at the CB2 receptor, virodhamine acted as a full agonist. Transport of [14 C]anandamide by RBL-2H3 cells was inhibited by virodhamine. Virodhamine produced hypothermia in the mouse and acted as an antagonist in the presence of anandamide both in vivo and in vitro. As a potential endogenous antagonist at the CB1 receptor, virodhamine adds a new form of regulation to the endocannabinoid system.Following the discovery of two G protein-coupled receptors, CB1 and CB2, which respond to ⌬ 9 -tetrahydrocannabinol, the active principal in marijuana, a search was initiated to identify endogenous ligands for these receptors. Anandamide (N-arachidonyl ethanolamide) was the first endocannabinoid discovered in 1992 by screening porcine brain extracts for compounds that bound to the cannabinoid receptor (Devane et al., 1992). It was later shown that anandamide could stimulate cannabinoid receptor-mediated signal transduction (Felder et al., 1993). The second endocannabinoid identified was 2-arachidonoyl glycerol (2-AG), which was isolated from canine gut and shown to have in vivo effects similar to ⌬ 9 -tetrahydrocannabinol (Mechoulam et al., 1995). Very recently, a third endocannabinoid, noladin ether, was also isolated from porcine brain (Hanus et al., 2001). Both anandamide and 2-AG are agonists at both the CB1 and CB2 receptors. Noladin ether has been shown to bind to the CB1 receptor with nanomolar affinity and to the CB2 receptor with low micromolar affinity, but functional activity has not yet been determined (Hanus et al., 2001).In the course of development of a bioanalytical method to measure anandamide in brain and peripheral tissues and brain microdialysate, a second analyte was seen that had the same molecular weight as anandamide but a shorter retention time, and therefore, could not be anandamide (Fig. 1). The peak was hypothesized to be O-arachidonoyl ethanolamine, and an authentic standard was subsequently synthesized (BIOMOL Research Laboratories, Plymouth Meeting, PA). Based on its chromatographic and mass spectrometric properties compared with the synthesized standard, the unknown analyte was confirme...

Fibroblast growth factor-21 (FGF-21) is a metabolic regulator that can influence glucose and lipid control in diabetic rodents and primates. We demonstrate that betaKlotho is an integral part of an activated FGF-21-betaKlotho-FGF receptor (FGFR) complex thus a critical subunit of the FGF-21 receptor. Cells lacking betaKlotho did not respond to FGF-21; the introduction of betaKlotho to these cells conferred FGF-21-responsiveness and recapitulated the entire scope of FGF-21 signaling observed in naturally responsive cells. Interestingly, FGF-21-mediated effects are heparin independent suggesting that betaKlotho plays a role in FGF-21 activity similar to the one played by heparin in the signaling of conventional FGFs. Moreover, in addition to conferring specificity for FGF-21, betaKlotho appears to support FGF-19 activity and mediates the receptor selectivity profile of FGF-19. All together, these results indicate that betaKlotho and FGFRs form the cognate FGF-21 receptor complex, mediating FGF-21 cellular specificity and physiological effects.

Pharmaceutical companies and regulatory agencies are broadly pursuing biomarkers as a means to increase the productivity of drug development. Quantifying differential levels of proteins from complex biological samples such as plasma or cerebrospinal fluid is one specific approach being used to identify markers of drug action, efficacy, toxicity, etc. We have developed a comprehensive, fully automated, and label-free approach to relative protein quantification from LC-MS/MS experiments of proteolytic protein digests including: de-noising, mass and charge state estimation, chromatographic alignment, and peptide quantification via integration of extracted ion chromatograms. Results from a variance components study of the entire method indicate that most of the variability is attributable to the LC-MS injection, with a median peptide LC-MS injection coefficient of variation of 8% on a ThermoFinnigan LTQ mass spectrometer. Spiked recovery results suggest a quantifiable range of approximately 32-fold for a sample protein.

AMPA-type glutamate receptors (GluRs) mediate most excitatory signaling in the brain and are composed of GluR principal subunits and transmembrane AMPA receptor regulatory protein (TARP) auxiliary subunits. Previous studies identified four mammalian TARPs, ␥-2 (or stargazin), ␥-3, ␥-4, and ␥-8, that control AMPA receptor trafficking, gating, and pharmacology. Here, we explore roles for the homologous ␥-5 and ␥-7 proteins, which were previously suggested not to serve as TARPs. Western blotting reveals high levels of ␥-5 and ␥-7 in the cerebellum, where ␥-7 is enriched in Purkinje neurons in the molecular layer and glomerular synapses in the granule cell layer. Immunoprecipitation proteomics shows that cerebellar ␥-7 avidly and selectively binds to AMPA receptor GluR subunits and also binds to the AMPA receptor clustering protein, postsynaptic density-95 (PSD-95). Furthermore, ␥-7 occurs together with PSD-95 and AMPA receptor subunits in purified postsynaptic densities. In heterologous cells, ␥-7 but not ␥-5 greatly enhances AMPA receptor glutamateevoked currents and modulates channel gating. In granule cells from stargazer mice, transfection of ␥-7 but not ␥-5 increases AMPA receptor-mediated currents. Compared with stargazin, ␥-7 differentially modulates AMPA receptor glutamate affinity and kainate efficacy. These studies define ␥-7 as a new member of the TARP family that can differentially influence AMPA receptors in cerebellar neurons.

Background:The recently discovered apolipoprotein A5 (ApoA5) is fast gaining attention as a key regulator of serum triglyceride concentrations. An ApoA5 mouse knock-out model produced an approximately fourfold increase in serum triglycerides, whereas a knock-in model with human ApoA5 produced 50 -70% lower concentrations of mouse serum triglycerides. In addition, peroxisome proliferator-activated receptor-␣ agonists, which are used clinically to lower serum triglyceride concentrations, cause increased ApoA5 mRNA expression. Despite these compelling molecular biology data, relatively little is known about ApoA5 protein in human serum. Methods: To better understand circulating concentrations and lipoprotein particle distribution of ApoA5, we expressed the recombinant human ApoA5 protein and raised antibodies against both the NH 2 and COOH termini. Results: Using the above reagents, we demonstrate for the first time that ApoA5 is present in human serum, although at much lower concentrations than other apolipoproteins such as ApoA1. Using a dual-antibody sandwich ELISA that we developed, we observed ApoA5 concentrations in human serum ranging from 24 to 406 g/L compared with ϳ1 g/L for ApoA1. We also

A robust top down proteomics method is presented for profiling alpha-synuclein species from autopsied human frontal cortex brain tissue from Parkinson's cases and controls. The method was used to test the hypothesis that pathology associated brain tissue will have a different profile of post-translationally modified alpha-synuclein than the control samples. Validation of the sample processing steps, mass spectrometry based measurements, and data processing steps were performed. The intact protein quantitation method features extraction and integration of m/z data from each charge state of a detected alpha-synuclein species and fitting of the data to a simple linear model which accounts for concentration and charge state variability. The quantitation method was validated with serial dilutions of intact protein standards. Using the method on the human brain samples, several previously unreported modifications in alpha-synuclein were identified. Low levels of phosphorylated alpha synuclein were detected in brain tissue fractions enriched for Lewy body pathology and were marginally significant between PD cases and controls (p = 0.03).

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