Inhibitors of V-ATPase proton transport reveal uncoupling functions of tether linking cytosolic and membrane domains of V0 subunit a (Vph1p).

Vacuolar ATPases (V-ATPases) are important for many cellular processes, as they regulate pH by pumping cytosolic protons into intracellular organelles. The cytoplasm is acidified when V-ATPase is inhibited; thus we conducted a high-throughput screen of a chemical library to search for compounds that acidify the yeast cytosol in vivo using pHluorin-based flow cytometry. Two inhibitors, alexidine dihydrochloride (EC(50) = 39 µM) and thonzonium bromide (EC(50) = 69 µM), prevented ATP-dependent proton transport in purified vacuolar membranes. They acidified the yeast cytosol and caused pH-sensitive growth defects typical of V-ATPase mutants (vma phenotype). At concentrations greater than 10 µM the inhibitors were cytotoxic, even at the permissive pH (pH 5.0). Membrane fractions treated with alexidine dihydrochloride and thonzonium bromide fully retained concanamycin A-sensitive ATPase activity despite the fact that proton translocation was inhibited by 80-90%, indicating that V-ATPases were uncoupled. Mutant V-ATPase membranes lacking residues 362-407 of the tether of Vph1p subunit a of V(0) were resistant to thonzonium bromide but not to alexidine dihydrochloride, suggesting that this conserved sequence confers uncoupling potential to V(1)V(0) complexes and that alexidine dihydrochloride uncouples the enzyme by a different mechanism. The inhibitors also uncoupled the Candida albicans enzyme and prevented cell growth, showing further specificity for V-ATPases. Thus, a new class of V-ATPase inhibitors (uncouplers), which are not simply ionophores, provided new insights into the enzyme mechanism and original evidence supporting the hypothesis that V-ATPases may not be optimally coupled in vivo. The consequences of uncoupling V-ATPases in vivo as potential drug targets are discussed.

Identification of potential human respiratory syncytial virus and metapneumovirus T cell epitopes using computational prediction and MHC binding assays.

Human respiratory syncytial virus (RSV) and human metapneumovirus (MPV) are two of the most common causes of serious viral lower respiratory tract illness in humans. CD8+ T cells have been shown to be important in animal models and human clinical studies for the clearance of viral infection, and they may contribute in part to protection against severe disease during reinfections. Precise enumeration and accurate phenotyping of RSV- or MPV-specific CD8+ T cells in humans is currently limited by the relatively small number of T cell epitopes that have been mapped with accompanying identification of MHC restriction patterns. We sought to expand the number of potential RSV and MPV epitopes for use in clinical and translational studies by identifying an expanded set of MHC-binding peptides based on RSV and MPV wild-type virus strain protein sequences. We interrogated the full protein sequences of all 9 or 11 proteins of MPV or RSV respectively using four established epitope prediction algorithms for human HLA A*0101, A*0201, or B*0702 binding and attempted to synthesize the top-scoring 150-152 peptides for each of the two viruses. Synthesis resulted in 442 synthesized and soluble peptides of the 452 predicted epitopes for MPV or RSV. We then determined the binding of the synthetic peptides to recombinant human HLA A*0101, A*0201 or B*0702 molecules with the predicted restriction using a commercially available plate-based assay, iTopia. A total of 230 of the 442 peptides tested exhibited binding to the appropriate MHC molecule. The binding results suggested that existing algorithms for prediction of MHC A*0201 binding are particularly robust. The binding results also provided a large benchmarking data collection for comparison of new prediction algorithms.

Comparison of competitive radioimmunoassays and two-site sandwich assays for the measurement and interpretation of plasma ghrelin levels.

CONTEXT: Ghrelin, an endogenous ligand for the GH secretagogue receptor, is an orexigenic peptide hormone produced primarily by the stomach. Recent studies suggest significant differences in the specificity of currently available ghrelin assays. OBJECTIVE: The aim of the study was to compare four ghrelin assays (two commercially available and two developed by our group) of differing specificity, each used on the same set of more than 800 plasma samples from a human study. DESIGN: Thirteen volunteers were sampled every 20 min for 6 h after consumption of one of three isocaloric drinks consisting of either 80% fat, 80% carbohydrate, or 80% protein. The samples were assayed by RIA for total and active ghrelin, as well as by sandwich assays for acyl and des-acyl ghrelin. The ghrelin profiles for each individual were smoothed using a statistical algorithm to lessen the effects of pulsatility and noise. RESULTS: The sandwich assays for acyl and des-acyl ghrelin yielded ghrelin values that were lower than those from the corresponding RIAs. The ghrelin profiles after nutrient ingestion were similar, yet key differences among the four assays were apparent; in particular, percentage changes were significantly greater in the sandwich assays. CONCLUSIONS: The lower levels and greater relative changes in ghrelin values reported by the sandwich assays are consistent with greater assay specificity. When applied to the nutrient study, the sandwich assays were better able to distinguish the different responses to different nutrients than were the RIAs.

Novel ghrelin assays provide evidence for independent regulation of ghrelin acylation and secretion in healthy young men.

CONTEXT: Ghrelin, an acylated peptide hormone secreted from the gut, regulates appetite and metabolism. Elucidating its pattern of secretion in the fed and fasted states is important in the face of the obesity epidemic. OBJECTIVE: Our objective was to examine changes in circulating ghrelin and des-acyl ghrelin in response to meals and fasting using newly developed two-site sandwich assays and sample preservation protocols to allow specific detection of full-length forms. DESIGN: Ten-minute sampling was done for 26.5 h during a fed admission with standardized meals and on a separate admission during the final 24 h of a 61.5-h fast and continuing for 2.5 h after terminating the fast. SETTING: The study was conducted at the University Hospital General Clinical Research Center. PARTICIPANTS: Eight male volunteers participated, mean +/- sd age 24.5 +/- 3.7 yr and body mass index 24 +/- 2.1 kg/m(2). MAIN OUTCOME MEASURES: Ten-minute sampling profiles were assessed for ghrelin and des-acyl ghrelin, fed and fasting. RESULTS: In the fed state, ghrelin and des-acyl ghrelin showed similar dynamics; both were sharply inhibited by meals and increased at night. During fasting, ghrelin decreased to nadir levels seen postprandially, and des-acyl ghrelin remained near peak levels seen preprandially. Total full-length ghrelin (acyl plus des-acyl) levels remained unchanged. CONCLUSIONS: Meals inhibited secretion of both ghrelin and des-acyl ghrelin, yet long-term fasting inhibited acylation but not total secretion. Acylation may be regulated independently of secretion by nutrient availability in the gut or by esterases that cleave the acyl group. These studies highlight the importance of stringent conditions for sample collection and evaluation of full-length ghrelin and des-acyl ghrelin using specific two-site assays.

Evidence for acyl-ghrelin modulation of growth hormone release in the fed state.

CONTEXT: The timing and frequency of GH secretory episodes is regulated by GHRH and somatostatin. This study provides evidence for amplification of these GH pulses by endogenous acyl-ghrelin. DESIGN: Blood was sampled every 10 min for 26.5 h during a fed admission with standardized meals and also during the final 24 h of a 61.5-h fast. GH secretion profiles were derived from deconvolution of 10-min sampling data, and full-length acyl-ghrelin levels were measured using a newly developed two-site sandwich assay. SETTING: The study was conducted at a university hospital general clinical research center. PARTICIPANTS: Participants included eight men with mean (+/- sd) age 24.5 +/- 3.7 yr (body mass index 24 +/- 2.1 kg/m(2)). RESULTS: Correlations were computed between amplitudes of individual GH secretory events and the average acyl-ghrelin concentration in the 60-min interval preceding each GH burst. In the fed state, the peak correlations were positive for all subjects and significantly higher than in the fasting state when acyl-ghrelin levels declined [mean (+/- sem): 0.7 (0.04) vs. 0.29 (0.08), P = 0.017]. In addition, long-term fasting was associated with an increase in the GH secretory pulse mass and amplitude but not frequency [fed vs. fasting pulse mass: 0.22 (0.05) vs. 0.44 (0.06) microg/liter, P = 0.002; amplitude: 5.2 (1.3) vs. 11.8 (1.9) microg/liter/min, P = 0.034; pulses per 24 h: 19.4 (0.5) vs. 22.0 (1.4), P = 0.1]. CONCLUSION: Our data support the hypothesis that under normal conditions in subjects given regular meals endogenous acyl-ghrelin acts to increase the amplitude of GH pulses.

Acyl and total ghrelin are suppressed strongly by ingested proteins, weakly by lipids, and biphasically by carbohydrates.

CONTEXT: Ghrelin is an orexigenic hormone that can increase body weight. Its circulating levels increase before meals and are suppressed after food ingestion. Understanding the effects of specific types of ingested macronutrients on ghrelin regulation could facilitate the design of weight-reducing diets. OBJECTIVE: We sought to understand how ingestion of carbohydrates, proteins, or lipids affect acyl (bioactive) and total ghrelin levels among human subjects, hypothesizing that lipids might suppress ghrelin levels less effectively than do either carbohydrates or proteins. DESIGN: This was a randomized, within-subjects cross-over study. SETTING: The study was conducted at a University Clinical Research Center. PARTICIPANTS: There were 16 healthy human subjects included in the study. INTERVENTIONS: Isocaloric, isovolemic beverages composed primarily of carbohydrates, proteins, or lipids were provided. MAIN OUTCOME MEASURES: The magnitude of postprandial suppression of total and acyl ghrelin levels (measured with a novel acyl-selective, two-site ELISA) was determined. RESULTS: All beverages suppressed plasma acyl and total ghrelin levels. A significant effect of macronutrient class on decremental area under the curve for both acyl and total ghrelin was observed; the rank order for magnitude of suppression was protein more than carbohydrate more than lipid. Total ghrelin nadir levels were significantly lower after both carbohydrate and protein, compared with lipid beverages. In the first 3 postprandial hours, the rank order for acyl and total ghrelin suppression was carbohydrate more than protein more than lipid. In the subsequent 3 h, there was a marked rebound above preprandial values of acyl and total ghrelin after carbohydrate ingestion alone. CONCLUSIONS: These findings suggest possible mechanisms contributing to the effects of high-protein/low-carbohydrate diets to promote weight loss, and high-fat diets to promote weight gain.