Positive Effect of Acetylation on Proteomic Analysis Based on Liquid Chromatography with Atmospheric Pressure Chemical Ionization and Photoionization Mass Spectrometry.

A typical bottom-up proteomic workflow comprises sample digestion with trypsin, separation of the hydrolysate using reversed-phase HPLC, and detection of peptides via electrospray ionization (ESI) tandem mass spectrometry. Despite the advantages and wide usage of protein identification and quantification, the procedure has limitations. Some domains or parts of the proteins may remain inadequately described due to inefficient detection of certain peptides. This study presents an alternative approach based on sample acetylation and mass spectrometry with atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). These ionizations allowed for improved detection of acetylated peptides obtained via chymotrypsin or glutamyl peptidase I (Glu-C) digestion. APCI and APPI spectra of acetylated peptides often provided sequence information already at the full scan level, while fragmentation spectra of protonated molecules and sodium adducts were easy to interpret. As demonstrated for bovine serum albumin, acetylation improved proteomic analysis. Compared to ESI, gas-phase ionizations APCI and APPI made it possible to detect more peptides and provide better sequence coverages in most cases. Importantly, APCI and APPI detected many peptides which passed unnoticed in the ESI source. Therefore, analytical methods based on chymotrypsin or Glu-C digestion, acetylation, and APPI or APCI provide data complementary to classical bottom-up proteomics.

Search for lipidized PrRP analogs with strong anorexigenic effect: In vitro and in vivo studies.

Prolactin-releasing peptide (PrRP) is an anorexigenic neuropeptide that attenuates food intake and increases energy expenditure. We designed three series of new lipidized PrRP31 analogs of different lengths of fatty acids attached at amino acids 1 or 11 directly or via linkers, part of them acetylated at the N-terminus and/or modified with dichlorophenylalanine (PheCl2) at the C-terminus. We tested their affinity for and activation of signaling pathways relevant to receptors GPR10, NPFF-R2, and NPFF-R1, effect on food intake in fasted or freely fed mice and rats, and stability in rat plasma. We aimed to select a strong dual GPR10/NPFF-R2 agonist whose affinity for NPFF-1 was not enhanced. The selected potent analog was then tested for body weight-lowering potency after chronic administration in mice with diet-induced obesity. PrRP31 analogs lipidized by monocarboxylic fatty acids showed strong dual affinity for both GPR10 and NPFF-R2 and activated MAPK/ERK1/2, Akt and CREB in cells overexpressing GPR10 and NPFF-R2. The selected analog stabilized at N- and C-termini and palmitoylated through the TTDS linker to Lys11 is a powerful dual agonist GPR10/NPFF-R2 at not enhanced affinity for NPFF-R1. It showed strong anti-obesity properties in mice with diet-induced obesity and became a potential compound for further studies.

A Novel Truncated Liver Enriched Antimicrobial Peptide-2 Palmitoylated at its N-Terminal Antagonizes Effects of Ghrelin.

Ghrelin is secreted in the stomach during fasting and targets the growth hormone secretagogue receptor (GHSR1a) in the hypothalamus and brainstem to exert its orexigenic effect. Recently, liver enriched antimicrobial peptide-2 (LEAP2) was identified as an endogenous high-affinity GHSR1a antagonist. LEAP2 is a 40-amino acid peptide with two disulfide bridges and GHRS1a affinity in the N-terminal hydrophobic part. In this study, we tested modified truncated N-terminal peptide LEAP2 (1-14), along with its myristoylated, palmitoylated, and stearoylated analogs, to determine their affinity to and activation of GHSR1a and their anorexigenic effects after acute peripheral administration. The lipidized analogs bound GHSR1a with affinity similar to that of natural LEAP2, and lipidization significantly enhanced the affinity of LEAP2(1-14) to GHSR1a. According to the beta-lactamase reporter gene response, the natural GHSR1a agonist ghrelin activated the receptor with nanomolar EC50 LEAP2(1-14) analogs behaved as inverse agonists of GHSR1a and suppressed internal activity of the receptor with EC50 values in the 10-8 M range. LEAP2(1-14) analogs significantly lowered acute food intake in overnight fasted mice, and palmitoylated LEAP2(1-14) was the most potent. In free-fed mice, all LEAP2(1-14) analogs significantly decreased the orexigenic effect of the stable ghrelin analog [Dpr3]Ghrelin. Moreover, palmitoylated LEAP2(1-14) inhibited the growth hormone (GH) release induced by [Dpr3] Ghrelin and exhibited an increased stability in rat plasma compared with LEAP2(1-14). In conclusion, palmitoylated LEAP2(1-14) had the most pronounced affinity for GHSR1a, had an anorexigenic effect, exhibited stability in rat plasma, and attenuated [Dpr3]Ghrelin-induced GH release. Such properties render palmitoylated LEAP2(1-14) a promising substance for antiobesity treatment. SIGNIFICANCE STATEMENT: The agonist and antagonist of one receptor are rarely found in one organism. For ghrelin receptor (growth hormone secretagogue receptor, GHSR), endogenous agonist ghrelin and endogenous antagonist/inverse agonist liver enriched antimicrobial peptide-2 (LEAP2) co-exist and differently control GHSR signaling. As ghrelin has a unique role in food intake regulation, energy homeostasis, and cytoprotection, lipidized truncated LEAP2 analogs presented in this study could serve not only to reveal the relationship between ghrelin and LEAP2 but also for development of potential anti-obesity agents.

In Vitro and In Vivo Characterization of Novel Stable Peptidic Ghrelin Analogs: Beneficial Effects in the Settings of Lipopolysaccharide-Induced Anorexia in Mice.

Ghrelin, the only known orexigenic gut hormone produced primarily in the stomach, has lately gained attention as a potential treatment of anorexia and cachexia. However, its biologic stability is highly limited; therefore, a number of both peptide and nonpeptide ghrelin analogs have been synthesized. In this study, we provide in vitro and in vivo characterization of a series of novel peptide growth hormone secretagogue receptor (GHS-R1a) agonists, both under nonpathologic conditions and in the context of lipopolysaccharide (LPS)-induced anorexia. These analogs were based on our previous series modified by replacing the Ser3 with diaminopropionic acid (Dpr), the N-terminal Gly with sarcosine, and Phe4 with various noncoded amino acids. New analogs were further modified by replacing the n-octanoyl bound to Dpr3 with longer or unsaturated fatty acid residues, by incorporation of the second fatty acid residue into the molecule, or by shortening the peptide chain. These modifications preserved the ability of ghrelin analogs to bind to the membranes of cells transfected with GHS-R1a, as well as the GHS-R1a signaling activation. The selected analogs exhibited long-lasting and potent orexigenic effects after a single s.c. administration in mice. The stability of new ghrelin analogs in mice after s.c. administration was significantly higher when compared with ghrelin and [Dpr3]ghrelin, with half-lives of approximately 2 hours. A single s.c. injection of the selected ghrelin analogs in mice with LPS-induced anorexia significantly increased food intake via the activation of orexigenic pathways and normalized blood levels of proinflammatory cytokines, demonstrating the anti-inflammatory potential of the analogs.

Impact of novel palmitoylated prolactin-releasing peptide analogs on metabolic changes in mice with diet-induced obesity.

Analogs of anorexigenic neuropeptides, such as prolactin-releasing peptide (PrRP), have a potential as new anti-obesity drugs. In our previous study, palmitic acid attached to the N-terminus of PrRP enabled its central anorexigenic effects after peripheral administration. In this study, two linkers, γ-glutamic acid at Lys11 and a short, modified polyethylene glycol at the N-terminal Ser and/or Lys11, were applied for the palmitoylation of PrRP31 to improve its bioavailability. These analogs had a high affinity and activation ability to the PrRP receptor GPR10 and the neuropeptide FF2 receptor, as well as short-term anorexigenic effect similar to PrRP palmitoylated at the N-terminus. Two-week treatment with analogs that were palmitoylated through linkers to Lys11 (analogs 1 and 2), but not with analog modified both at the N-terminus and Lys11 (analog 3) decreased body and liver weights, insulin, leptin, triglyceride, cholesterol and free fatty acid plasma levels in a mouse model of diet-induced obesity. Moreover, the expression of uncoupling protein-1 was increased in brown fat suggesting an increase in energy expenditure. In addition, treatment with analogs 1 and 2 but not analog 3 significantly decreased urinary concentrations of 1-methylnicotinamide and its oxidation products N-methyl-2-pyridone-5-carboxamide and N-methyl-4-pyridone-3-carboxamide, as shown by NMR-based metabolomics. This observation confirmed the previously reported increase in nicotinamide derivatives in obesity and type 2 diabetes mellitus and the effectiveness of analogs 1 and 2 in the treatment of these disorders.

Novel approach to determine ghrelin analogs by liquid chromatography with mass spectrometry using a monolithic column.

In our project, ghrelin analogs possessing enhanced stability and potential to significantly increase food intake were used. Three newly synthesized ghrelin analogs with fatty acid residues consisting of 8, 10, and 14 carbon atoms were studied. The main goal of this work was to develop a suitable analytical method for the determination of the stability of the novel ghrelin analogs in plasma. An appropriate liquid chromatography-mass spectrometry method was developed and optimized. The results obtained were compared with the data measured by using a commercial enzyme-linked immunosorbent assay kit, and a good correlation was found. A preparation strategy for plasma samples was optimized and consisted of simple dilution of the plasma samples followed by direct injection onto a very short monolithic column in combination with mass spectrometric detection. The developed analytical method was utilized for the determination of the stability of the prepared lipopeptides in plasma and for the quantification of the lipopeptides in a preliminary pharmacokinetic study. The feasibility of the developed separation method was clearly demonstrated. Accuracy and precision were within 80-120% and ±20% limits, respectively. Calibration curves were constructed in the range of 1-250 µg/mL.

Glutamate carboxypeptidase II does not process amyloid-ß peptide.

The accumulation of amyloid-ß (Aß) peptide is thought to be a major causative mechanism of Alzheimer's disease. Aß accumulation could be caused by dysregulated processing of amyloid precursor protein, yielding excessive amounts of Aß, and/or by inefficient proteolytic degradation of the peptide itself. Several proteases have been described as Aß degradation enzymes, most notably metalloendopeptidases, aspartic endopeptidases, and some exopeptidases. Recently a report suggested that another metallopeptidase, glutamate carboxypeptidase II (GCPII), can also cleave Aß. GCPII is a zinc exopeptidase that cleaves glutamate from N-acetyl-L-aspartyl-L-glutamate in the central nervous system and from pteroylpoly-γ-glutamate in the jejunum. GCPII has been proposed as a promising therapeutic target for disorders caused by glutamate neurotoxicity. However, an Aß-degrading activity of GCPII would compromise potential pharmaceutical use of GCPII inhibitors, because the enzyme inhibition might lead to increased Aß levels and consequently to Alzheimer's disease. Therefore, we analyzed the reported Aß-degrading activity of GCPII using highly purified recombinant enzyme and synthetic Aß. We did not detect any Aß degradation activity of GCPII or its homologue even under prolonged incubation at a high enzyme to substrate ratio. These results are in good agreement with the current detailed structural understanding of the substrate specificity and enzyme-ligand interactions of GCPII.

Neuropeptide FF analog RF9 is not an antagonist of NPFF receptor and decreases food intake in mice after its central and peripheral administration.

Neuropeptide FF (NPFF) belongs to the RF-amide family of peptides bearing the identical C-terminal amino acid sequence (R-F-NH2). In addition to NPFF, prolactin-releasing peptide (PrRP), another RF-amide, binds to NPFF receptors with high affinity. A selective antagonist of PrRP has not yet been identified, but a selective antagonist of NPFF, 1-adamantanecarbonyl-RF-NH2 (RF9), was recently reported to antagonize the hyperalgesic effect of NPFF after central administration to mice. In the present study, RF9 competed with NPFF analog D-Y-L-(N-Me)-F-Q-P-Q-R-F-NH2 (1DMe) in binding to CHO-K1 cell membranes transfected with the human NPFF2 receptor. In rat pituitary RC-4B/C cells, where the expression of the NPFF2 receptor was proved by immunodetection, RF9 did not reverse the phosphorylation of MAPK/ERK1/2 induced by [Tyr(1)]NPFF. In vivo experiments with fasted mice confirmed that centrally injected [Tyr(1)]NPFF significantly lowered food intake. However, RF9, a putative NPFF2 antagonist, did not reverse the anorectic effect of [Tyr(1)]NPFF. Paradoxically, RF9 itself exhibited an anorectic effect in fasted mice not only after intracerebroventricular but also after subcutaneous administration. This finding casts doubt on claims that RF9 is an NPFF antagonist.

New analogs of the CART peptide with anorexigenic potency: the importance of individual disulfide bridges.

The CART (cocaine- and amphetamine-regulated transcript) peptide is an anorexigenic neuropeptide that acts in the hypothalamus. The receptor and the mechanism of action of this peptide are still unknown. In our previous study, we showed that the CART peptide binds specifically to PC12 rat pheochromocytoma cells in both the native and differentiated into neuronal phenotype. Two biologically active forms, CART(55-102) and CART(61-102), with equal biological activity, contain three disulfide bridges. To clarify the importance of each of these disulfide bridges in maintaining the biological activity of CART(61-102), an Ala scan at particular S-S bridges forming cysteines was performed, and analogs with only one or two disulfide bridges were synthesized. In this study, a stabilized CART(61-102) analog with norleucine instead of methionine at position 67 was also prepared and was found to bind to PC12 cells with an anorexigenic potency similar to that of CART(61-102). The binding study revealed that out of all analogs tested, [Ala(68,86)]CART(61-102), which contains two disulfide bridges (positions 74-94 and 88-101), preserved a high affinity to both native PC12 cells and those that had been differentiated into neurons. In food intake and behavioral tests with mice after intracerebroventricular administration, this analog showed strong and long-lasting anorexigenic potency. Therefore, the disulfide bridge between cysteines 68 and 86 in CART(61-102) can be omitted without a loss of biological activity, but the preservation of two other disulfide bridges and the full-length peptide are essential for biological activity.

Characterization of new stable ghrelin analogs with prolonged orexigenic potency.

Ghrelin, the only known peripherally produced and centrally acting peptide that stimulates food intake, is synthesized primarily in the stomach and acts through the growth hormone secretagogue receptor (GHS-R1a). In addition to its orexigenic effect, ghrelin stimulates the release of growth hormone (GH). In this study, we investigated the biological properties of full-length and shortened ghrelin analogs in which octanoylated Ser(3) is replaced with an octanoic acid moiety coupled to diaminopropionic acid (Dpr). Ghrelin analogs stabilized with Dpr(N-octanoyl) in position 3 and noncoded amino acids in position 1 (sarcosine) and/or position 4 (naphthylalanine or cyclohexylalanine) were found to possess affinities similar to those of ghrelin for cell membranes with transfected GHS-R1a. In vivo, the prolonged orexigenic effects of analogs containing Dpr(N-octanoyl)(3) compared with that of ghrelin in adult mice and a similar impact on GH secretion in young mice were found. Full-length [Dpr(N-octanoyl)(3)]ghrelin and its analogs with a noncoded amino acid in position 1 and/or 4 showed significantly prolonged stability in blood plasma compared with that of ghrelin. Ghrelin analogs with a prolonged orexigenic effect are potential treatments for GH deficiency or cachexia that accompanies chronic diseases. Desoctanoylated ghrelin analogs and N-terminal penta- and octapeptides of ghrelin did not show any biological activity.

Biological properties of prolactin-releasing peptide analogs with a modified aromatic ring of a C-terminal phenylalanine amide.

Prolactin-releasing peptide (PrRP)-induced secretion of prolactin is not currently considered a primary function of PrRP, but the development of late-onset obesity in both PrRP and PrRP receptor knock-out mice indicates the unique anorexigenic properties of PrRP. In our recent study, we showed comparable potencies of peptides PrRP31 and PrRP20 in binding, intracellular signaling and prolactin release in pituitary RC-4B/C cells, and anorexigenic effect after central administration in fasted mice. In the present study, eight analogs of PrRP20 with C-terminal Phe amide modified with a bulky side chain or a halogenated aromatic ring revealed high binding potency, activation of mitogen-activated protein kinase/extracellular-regulated kinase (MAPK/ERK1/2) and cAMP response element-binding protein (CREB) and prolactin release in RC-4B/C cells. In particular, [PheNO(2)(31)]PrRP20, [1-Nal(31)]PrRP20, [2-Nal(31)]PrRP20 and [Tyr(31)]PrRP20 showed not only in vitro effects comparable or higher than those of PrRP20, but also a very significant and long-lasting anorexigenic effect after central administration in fasted mice. The design of potent and long-lasting PrRP analogs with selective anorexigenic properties promises to contribute to the study of food intake disorders.

Characterization of prolactin-releasing peptide: binding, signaling and hormone secretion in rodent pituitary cell lines endogenously expressing its receptor.

The recently discovered prolactin-releasing peptide (PrRP) binds to the PrRP receptor and is involved in endocrine regulation and energy metabolism. However, its main physiological role is currently unknown. Two biologically active isoforms of PrRP exist: the 31 (PrRP31) and the 20 (PrRP20) amino acid forms, which both contain a C-terminal Phe amide sequence. In the present study, the PrRP receptor was immunodetected in three rodent tumor pituitary cell lines: GH3, AtT20 and RC-4B/C cells. The saturation binding of radioiodinated PrRP31 to intact cells demonstrated a K(d) in the 10(-9)M range and a B(max) in the range of tens of thousands binding sites per cell. For binding to RC-4B/C cells, both PrRP31 and PrRP20 competed with (125)I-PrRP31 with a similar K(i). The C-terminal analog PrRP13 showed lower binding potency compared to PrRP31 and PrRP20. All PrRP analogs increased the phosphorylation of MAPK/ERK1/2 (mitogen-activated phosphorylase/extracellular-regulated kinase) and CREB (cAMP response element-binding protein) in RC-4B/C cells. Additionally, prolactin release was induced by the PrRP analogs in a dose-dependent manner in RC-4B/C cells. Finally, food intake after intracerebroventricular administration of PrRP analogs in fasted mice was followed. Both PrRP31 and PrRP20 decreased food intake, but PrRP13 did not show significant effect. Studies on pituitary cell lines expressing the PrRP receptor are more physiologically relevant than those on cells transfected with the receptor. This cell type can be used as a model system for pharmacological studies searching for PrRP antagonists and stable effective PrRP agonists, as these drugs may have potential as anti-obesity agents.

A new epigenetic marker: the replication-coupled, cell cycle-dependent, dual modification of the histone H4 tail.

Recently, we described the cold-dependent detection of an epitope, epiC, that was selectively recognized by a monoclonal anti-actin antibody at 4 degrees C, but not at RT, in the early replicating chromatin domains of human fibroblast cell nuclei and chromosomes. EpiC was present in a distinct cell cycle window extending from S-phase throughout mitosis until early G1-phase of the next cell generation, indicating its possible involvement in the transfer/maintenance of epigenetic information on transcriptionally competent parts of the genome. However, the molecular nature of epiC remained unresolved. Here we identified epiC as a dual post-translational modification on the same histone H4 tail, which was immunodetected for the first time. We show that the antibody selectively recognized a synthetic peptide of the histone H4 region K12-L22 containing acetylated K16 and dimethylated K20 (H4K16ac-K20me2) at 4 degrees C, but not at RT. Moreover, we show that the peptide containing acetylated K16 and either unmodified or monomethylated K20 was recognized by this antibody at both temperatures. The present and previous results together indicate that, by acetylation of histone H4 K16 during S-phase, the early replicating chromatin domains acquire the H4K16ac-K20me2 epigenetic label that persists on the chromatin throughout mitosis and become deacetylated during early G1-phase of the next cell cycle.

Structure-activity relationship of CART (cocaine- and amphetamine-regulated transcript) peptide fragments.

CART (cocaine- and amphetamine-regulated transcript) peptides are neuropeptides abundant in the central nervous system and periphery found to be involved in the regulation of food intake behavior and other physiological processes. Recently, we reported specific binding of (125)I-CART(61-102) to the rat adrenal pheochromocytoma cell line PC12, both intact cells and cell membranes. In this study, several fragments of CART(61-102) corresponding to its structural loops were synthesized and tested for their potency in binding experiments using PC12 intact cells and cell membranes and in feeding test with fasted mice. From all shorter peptides tested, only CART(74-86) and CART(62-86) containing disulfide bridges kept partial binding potency of the original molecule with K(i) in 10(-5) and 10(-4)M range. However, these fragments were not able to inhibit food intake after their central administration up to a dose of 4 nmol/mouse. The results showed that a compact structure containing three disulfide bridges is necessary for preservation of full biological activity of CART peptides.

Biolocalisation and photochemical properties of two novel macrocyclic photosensitisers: a spectroscopic study.

Biolocalisation and photochemical properties of novel macrocyclic photosensitisers, guanidiniocarbonyl-substituted tetraphenylporphyrin (1) and sugar-substituted sapphyrin (2) were investigated by spectroscopic methods. Both photosensitisers absorb in far visible region and showed good tumour localisation. Photosensitiser 2 demonstrated significantly larger absolute and relative to normal tissue (T/N) amount in tumour (330 microg g(-1) wet tissue, T/N=19.0) than photosensitiser 1 did (13 microg g(-1) wet tissue, T/N=2.1). According to iodometric and uric acid assays, compound 1 produced large amount of 1O2 (phidelta=0.60-0.68), while compound 2 showed non-significant 1O2 production (phidelta=0.04). The electronic spectroscopic study confirms that only photosensitiser 1 is able to mediate photooxidation of model compounds (BSA, poly(Trp), Tyr, Trp, and GMP) after light irradiation. Pour photochemical activity of compound 2 was explained by its self-aggregation. Raman spectroscopic study indicated that monomerised photosensitiser 2 effectively damaged BSA and calf thymus DNA after light excitation at the conditions of high excess of these macromolecules.