Investigating endogenous peptides and peptidases using peptidomics

Rather than simply being protein degradation products, peptides have proven to be important bioactive molecules. Bioactive peptides act as hormones, neurotransmitters, and antimicrobial agents in vivo. The dysregulation of bioactive peptide signaling is also known to be involved in disease, and targeting peptide hormone pathways has been a successful strategy in the development of novel therapeutics. The importance of bioactive peptides in biology has spurred research to elucidate the function and regulation of these molecules. Classical methods for peptide analysis have relied on targeted immunoassays, but certain scientific questions necessitated a broader and more detailed view of the peptidome--all the peptides in a cell, tissue, or organism. In this review we discuss how peptidomics has emerged to fill this need through the application of advanced liquid chromatography--tandem mass spectrometry (LC-MS/MS) methods that provide unique insights into peptide activity and regulation.     

Comparative peptidomics of Caenorhabditis elegans versus C. briggsae by LC-MALDI-TOF MS

Neuropeptides are important signaling molecules that function in cell-cell communication as neurotransmitters or hormones to orchestrate a wide variety of physiological conditions and behaviors. These endogenous peptides can be monitored by high throughput peptidomics technologies from virtually any tissue or organism. The neuropeptide complement of the soil nematode Caenorhabditis elegans has been characterized by on-line two-dimensional liquid chromatography and quadrupole time-of-flight tandem mass spectrometry (2D-nanoLC Q-TOF MS/MS). Here, we use an alternative peptidomics approach combining liquid chromatography (LC) with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry to map the peptide content of C. elegans and another Caenorhabditis species, Caenorhabditis briggsae. This study allows a better annotation of neuropeptide-encoding genes from the C. briggsae genome and provides a promising basis for further evolutionary comparisons.     

Disrupted circadian rhythms in VIP- and PHI-deficient mice

The related neuropeptides vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI) are expressed at high levels in the neurons of the suprachiasmatic nucleus (SCN), but their function in the regulation of circadian rhythms is unknown. To study the role of these peptides on the circadian system in vivo, a new mouse model was developed in which both VIP and PHI genes were disrupted by homologous recombination. In a light-dark cycle, these mice exhibited diurnal rhythms in activity which were largely indistinguishable from wild-type controls. In constant darkness, the VIP/PHI-deficient mice exhibited pronounced abnormalities in their circadian system. The activity patterns started approximately 8 h earlier than predicted by the previous light cycle. In addition, lack of VIP/PHI led to a shortened free-running period and a loss of the coherence and precision of the circadian locomotor activity rhythm. In about one-quarter of VIP/PHI mice examined, the wheel-running rhythm became arrhythmic after several weeks in constant darkness. Another striking example of these deficits is seen in the split-activity patterns expressed by the mutant mice when they were exposed to a skeleton photoperiod. In addition, the VIP/PHI-deficient mice exhibited deficits in the response of their circadian system to light. Electrophysiological analysis indicates that VIP enhances inhibitory synaptic transmission within the SCN of wild-type and VIP/PHI-deficient mice. Together, the observations suggest that VIP/PHI peptides are critically involved in both the generation of circadian oscillations as well as the normal synchronization of these rhythms to light.     

Snapin mediates incretin action and augments glucose-dependent insulin secretion

Impaired insulin secretion contributes to the pathogenesis of type 2 diabetes mellitus (T2DM). Treatment with the incretin hormone glucagon-like peptide-1 (GLP-1) potentiates insulin secretion and improves metabolic control in humans with T2DM. GLP-1 receptor-mediated signaling leading to insulin secretion occurs via cyclic AMP stimulated protein kinase A (PKA)- as well as guanine nucleotide exchange factor-mediated pathways. However, how these two pathways integrate and coordinate insulin secretion remains poorly understood. Here we show that these incretin-stimulated pathways converge at the level of snapin, and that PKA-dependent phosphorylation of snapin increases interaction among insulin secretory vesicle-associated proteins, thereby potentiating glucose-stimulated insulin secretion (GSIS). In diabetic islets with impaired GSIS, snapin phosphorylation is reduced, and expression of a snapin mutant, which mimics site-specific phosphorylation, restores GSIS. Thus, snapin is a critical node in GSIS regulation and provides a potential therapeutic target to improve β cell function in T2DM.     

Prolactin-releasing activity of porcine intestinal peptide (PHI-27)

Porcine intestinal peptide (PHI), a twenty-seven amino acid peptide isolated from porcine gut extracts, is a close structural homolog of the secretin family hormones. The structural and biological similarities of PHI to vasoactive intestinal peptide (VIP) together with its presence in the rat hypothalamus suggested a possible role for the peptide in the control of prolactin (PRL) secretion. PHI induced significant, dose-related stimulations of PRL release from cultured, dispersed rat pituitary cells in vitro. The minimum effective dose is 10⁷ molar, compared to 10⁹ molar for VIP. No interactive effect with thyrotropin-releasing hormone was observed; however, PHI partially overcame the dopamine inhibition of PRL release.     

Prkar1a in the regulation of insulin secretion

The incidence of type 2 diabetes mellitus (T2DM) is rapidly increasing worldwide with significant consequences on individual quality of life as well as economic burden on states' healthcare costs. While origins of the pathogenesis of T2DM are poorly understood, an early defect in glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is considered a hallmark of T2DM 1.Upon a glucose stimulus, insulin is secreted in a biphasic manner with an early first-phase burst of insulin, which is followed by a second, more sustained phase of insulin output 2. First phase insulin secretion is diminished early in T2DM as well is in subjects who are at risk of developing T2DM 3 4 5 6.An effective treatment of T2DM with incretin hormone glucagon-like peptide-1 (GLP-1) or its long acting peptide analogue exendin-4 (E4), restores first-phase and augments second-phase glucose stimulated insulin secretion. This effect of incretin action occurs within minutes of GLP-1/E4 infusion in T2DM humans. An additional important consideration is that incretin hormones augment GSIS only above a certain glucose threshold, which is slightly above the normal glucose range. This ensures that incretin hormones stimulate GSIS only when glucose levels are high, while they are ineffective when insulin levels are below a certain threshold 7 8.Activation of the GLP-1 receptor, which is highly expressed on pancreatic β-cells, stimulates 2 -distinct intracellular signaling pathways: a) the cAMP-protein kinase A branch and b) the cAMP-EPAC2 (EPAC=exchange protein activated by cAMP) branch. While the EPAC2 branch is considered to mediate GLP-1 effects on first-phase GSIS, the PKA branch is necessary for the former branch to be active 9 10. However, how these 2 branches interplay and converge and how their effects on insulin secretion and insulin vesicle exocytosis are coordinated is poorly understood.Thus, at the outset of our studies we have a poorly understood intracellular interplay of cAMP-dependent signaling pathways, which - when stimulated - restore glucose-dependent first phase and augment second phase insulin secretion in the ailing β-cells of T2DM.     

Identification of multiple antimicrobial peptides from the skin of fine-spined frog, Hylarana spinulosa (Ranidae)

In this study, peptidomics and genomics analyses were used to study antimicrobial peptides from the skin of Hylarana spinulosa. Twenty-nine different antimicrobial peptide precursors were characterized from the skin of H. spinulosa, which produce 23 mature antimicrobial peptides belonging to 12 different families. To confirm the actual presence and characteristics of these antimicrobial peptides in the skin tissue extractions from H. spinulosa, we used two distinct methods, one was peptide purification method that combined gel filtration chromatography and reversed-phase high performance liquid chromatography (RP-HPLC), and the other was peptidomics approach based on liquid chromatography in conjunction with tandem mass spectrometry (LC–MS/MS). In the peptidomics approach, incomplete tryptic digestion and gas-phase fractionation (GPF) analysis were used to increase peptidome coverage and reproducibility of peptide ion selection. Multiple species of microorganisms were chosen to test and analyze the antimicrobial activities and spectrum of these antimicrobial peptides.     

High-performance liquid chromatography and studies of neurophysin—neurohypophysial hormone pathways

High-performance liquid chromatography (HPLC) is being used extensively to characterize active polypeptides, precursor processing mechanisms, and cooperative peptide—protein noncovalent complexes in neuroendocrine pathways for neurohypophysial peptide hormones, oxytocin and vasopressin, and the hormone-associated proteins, neurophysins. Reversed-phase and ion-exchange HPLC polypeptide mapping have been used to detect the hormones, associated proteins, and other molecular forms containing these. This mapping but also ultimately to identify anatomical sites which contain the neurophysin/ hormone molecular pathways and to define the relatedness of polypeptide forms contained in different pathways. Reversed-phase HPLC also has provided a means to study proteolytic precursor processing, both to isolate synthetic and semisynthetic polypeptides and intermediates produced by these reactions. Finally, bioaffinity HPLC is being evaluated as a separatory and analytical tool. The latter includes its use to characterize the noncovalent peptide—protein and protein—protein interactions which occur among the molecular forms of the neurophysin/hormone pathways. These experiments typify the impact of HPLC for both analytical and preparative separations in studies of biologically active peptides and proteins.     

Cathepsin L and Arg/Lys aminopeptidase: a distinct prohormone processing pathway for the biosynthesis of peptide neurotransmitters and hormones

Peptide neurotransmitters and hormones are synthesized as protein precursors that require proteolytic processing to generate smaller, biologically active peptides that are secreted to mediate neurotransmission and hormone actions. Neuropeptides within their precursors are typically flanked by pairs of basic residues, as well as by monobasic residues. In this review, evidence for secretory vesicle cathepsin L and Arg/Lys aminopeptidase as a distinct proteolytic pathway for processing the prohormone proenkephalin is presented. Cleavage of prohormone processing sites by secretory vesicle cathepsin L occurs at the NH2-terminal side of dibasic residues, as well as between the dibasic residues, resulting in peptide intermediates with Arg or Lys extensions at their NH2-termini. A subsequent Arg/Lys aminopeptidase step is then required to remove NH2-terminal basic residues to generate the final enkephalin neuropeptide. The cathepsin L and Arg/Lys aminopeptidase prohormone processing pathway is distinct from the proteolytic pathway mediated by the subtilisin-like prohormone convertases 1/3 and 2 (PC1/3 and PC2) with carboxypeptidase E/H. Differences in specific cleavage sites at paired basic residue sites distinguish these two pathways. These two proteolytic pathways demonstrate the increasing complexity of regulatory mechanisms for the production of peptide neurotransmitters and hormones.     

Correlation-associated peptide networks of human cerebrospinal fluid

Profiling of peptides and small proteins from either human body fluids or tissues by chromatography and subsequent mass spectrometry reveals several thousand individual peptide signals per sample. Any peptide is an intermediate in the course of biosynthesis, post-translational modification (PTM), proteolytic processing and degradation. Changes in the concentration of one peptide often affects the concentration of the other, hence a challenge consists in the development of suitable tools to turn this large amount of data into biologically relevant information. Comprehensive statistical analysis of the peptide profiling data allows associating peptides, which are closely related in terms of peptide biochemistry. Here, the bioinformatic concept of peptide networks, correlation-associated peptide networks (CANs), is introduced. Peptides with statistical similarity of their concentrations are grouped in form of networks, and these networks are interpreted in terms of peptide biochemistry. The spectrum of functional relationships found in cerebrospinal fluid CAN covers PTM and proteolytic degradation of peptides, clearance processing in the complement cascade, common secretion of peptides by neuroendocrine cells as well as ubiquitin-mediated degradation. Our results indicate that CAN is a powerful bioinformatic tool for the systematic analysis and interpretation of large peptidomics and proteomics data and helps to discover novel bioactive and diagnostic peptides.     

Comparison of the insulinotropic activity of glucagon-superfamily peptides in rat pancreas perfusion.

Previous studies have demonstrated that glucagon-superfamily peptides stimulate insulin release from the pancreatic islets in a glucose dependent manner. In this study we have carried out a structure-activity study of their insulinotropic activity using a rat pancreas perfusion with 5.5 mM glucose concentration. The following peptides were examined: glucagon-like peptide-1(7-36)amide (tGLP-1), glucagon, gastric inhibitory peptide (GIP), peptide having an amino-terminal histidine and carboxy-terminal isoleucine amide (PHI), vasoactive intestinal polypeptide (VIP), growth hormone releasing factor(1-29)amide (GRF), GRF(1-27)amide and synthetic hybrid-peptides of PHI-GRF, PHI(1-11)-GRF(12-27) and PHI(1-20)-GRF(21-27). Their potencies were evaluated as: tGLP-1 = GIP > glucagon > PHI = VIP > PHI(1-20)-GRF(21-27) > PHI(1-11)-GRF(12-27) > GRF(1-29) = GRF(1-27). It is clear that 0.1 nM tGLP-1 stimulated insulin release, whereas 1 microM GRF(1-29) did not. These results indicate that 1) in addition to N-terminal amino acid (histidine or tyrosine), position 4 (glycine), position 9 (aspartic acid) and position 11 (serine) in the amino acid sequence are important for their insulinotropic activity, 2) not only the N-terminal portion but also the C-terminal portion of these peptides contribute to their insulinotropic activity.     

amontillado,the Drosophila homolog of the prohormone processing protease PC2, is required during embryogenesis and early larval development

Biosynthesis of most peptide hormones and neuropeptides requires proteolytic excision of the active peptide from inactive proprotein precursors, an activity carried out by subtilisin-like proprotein convertases (SPCs) in constitutive or regulated secretory pathways. The Drosophila amontillado (amon) gene encodes a homolog of the mammalian PC2 protein, an SPC that functions in the regulated secretory pathway in neuroendocrine tissues. We have identified amon mutants by isolating ethylmethanesulfonate (EMS)-induced lethal and visible mutations that define two complementation groups in the amon interval at 97D1 of the third chromosome. DNA sequencing identified the amon complementation group and the DNA sequence change for each of the nine amon alleles isolated. amon mutants display partial embryonic lethality, are defective in larval growth, and arrest during the first to second instar larval molt. Mutant larvae can be rescued by heat-shock-induced expression of the amon protein. Rescued larvae arrest at the subsequent larval molt, suggesting that amon is also required for the second to third instar larval molt. Our data indicate that the amon proprotein convertase is required during embryogenesis and larval development in Drosophila and support the hypothesis that AMON acts to proteolytically process peptide hormones that regulate hatching, larval growth, and larval ecdysis.     

Stimulatory effect of peptide histidine isoleucine amide 1-27 on prolactin release in the rat

Intravenous injection of pure peptide histidine isoleucine amide 1-27 (PHI) resulted in a prompt and significant increase of plasma prolactin (PRL) in conscious freely-moving male rats. Using a perifusion system of rat anterior pituitary tissues in vitro, effluent PRL levels were also increased by 10(-8)-10(-7) M PHI. A PRL releasing potency of PHI was almost similar with that of vasoactive intestinal polypeptide (VIP) or TRH both in vivo and in vitro. Coupled with the recent immunocytochemical studies showing the dense network of PHI immunoreactive fibers around the hypophysial portal vessels, PHI might be another candidate for PRL releasing factor.     

Peptidomics analysis of neuropeptides involved in copulatory behavior of the mollusk Lymnaea stagnalis

Male copulation behavior in mollusks is controlled by an array of peptide messengers. In the present study, we have used a peptidomics approach employing liquid chromatography in conjunction with electrospray mass spectrometry to characterize peptides contained in the penial complex of the freshwater snail, Lymnaea stagnalis. In addition to the previously described peptides, we have identified a group of novel peptides that share the carboxyl termini of -FVRIamide. A cDNA cloning study revealed the organization of the precursor, which contains 20 peptide domains with the carboxyl termini of -F(X)RIamide which are flanked by many putative proteolytic sites including the KR and the less commonly occurring (G)K and (G)R sites. In addition, there are several monobasic R and dibasic RR and KK sites that may be used for processing. We then used MALDI-TOF/TOF-MS in a data-dependent mode, which selected all the molecular ion species with the predicted masses of the mature -F(X)RIamide peptides, and performed MS/MS analysis on these peptides. This approach allowed us to identify all the predicted -F(X)RIamide peptides. Immunocytochemistry showed the localization of -FVRIamide immunoreactive neurons in several central ganglia, and immunoreactive axons in the penial complex. Finally, application of synthetic -FVRIamide peptides to an in vitro posterior vas deferens preparation showed inhibitory effect on the spontaneous contraction/relaxation cycle of the vas deferens.     

A pyruvate cycling pathway involving cytosolic NADP-dependent isocitrate dehydrogenase regulates glucose-stimulated insulin secretion

Glucose-stimulated insulin secretion (GSIS) from pancreatic islet beta-cells is central to control of mammalian fuel homeostasis. Glucose metabolism mediates GSIS in part via ATP-regulated K+ (KATP) channels, but multiple lines of evidence suggest participation of other signals. Here we investigated the role of cytosolic NADP-dependent isocitrate dehydrogenase (ICDc) in control of GSIS in beta-cells. Delivery of small interfering RNAs specific for ICDc caused impairment of GSIS in two independent robustly glucose-responsive rat insulinoma (INS-1-derived) cell lines and in primary rat islets. Suppression of ICDc also attenuated the glucose-induced increments in pyruvate cycling activity and in NADPH levels, a predicted by-product of pyruvate cycling pathways, as well as the total cellular NADP(H) content. Metabolic profiling of eight organic acids in cell extracts revealed that suppression of ICDc caused increases in lactate production in both INS-1-derived cell lines and primary islets, consistent with the attenuation of pyruvate cycling, with no significant changes in other intermediates. Based on these studies, we propose that a pyruvate cycling pathway involving ICDc plays an important role in control of GSIS.     

AMP-activated protein kinase plays a role in the control of food intake

AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade that acts as an intracellular energy sensor maintaining the energy balance within the cell. The finding that leptin and adiponectin activate AMPK to alter metabolic pathways in muscle and liver provides direct evidence for this role in peripheral tissues. The hypothalamus is a key regulator of food intake and energy balance, coordinating body adiposity and nutritional state in response to peripheral hormones, such as leptin, peptide YY-(3-36), and ghrelin. To date the hormonal regulation of AMPK in the hypothalamus, or its potential role in the control of food intake, have not been reported. Here we demonstrate that counter-regulatory hormones involved in appetite control regulate AMPK activity and that pharmacological activation of AMPK in the hypothalamus increases food intake. In vivo administration of leptin, which leads to a reduction in food intake, decreases hypothalamic AMPK activity. By contrast, injection of ghrelin in vivo, which increases food intake, stimulates AMPK activity in the hypothalamus. Consistent with the effect of ghrelin, injection of 5-amino-4-imidazole carboxamide riboside, a pharmacological activator of AMPK, into either the third cerebral ventricle or directly into the paraventricular nucleus of the hypothalamus significantly increased food intake. These results suggest that AMPK is regulated in the hypothalamus by hormones which regulate food intake. Furthermore, direct pharmacological activation of AMPK in the hypothalamus is sufficient to increase food intake. These findings demonstrate that AMPK plays a role in the regulation of feeding and identify AMPK as a novel target for anti-obesity drugs.     

Profiling thimet oligopeptidase-mediated proteolysis in Arabidopsis thaliana

Protein homeostasis (proteostasis) is crucial for proper cellular function, including the production of peptides with biological functions through controlled proteolysis. Proteostasis has roles in maintenance of cellular functions and plant interactions with the environment under physiological conditions. Plant stress continues to reduce agricultural yields causing substantial economic losses; thus, it is critical to understand how plants perceive stress signals to elicit responses for survival. As previously shown in Arabidopsis thaliana, thimet oligopeptidases (TOPs) TOP1 (also referred to as organellar oligopeptidase) and TOP2 (also referred to as cytosolic oligopeptidase) are essential components in plant response to pathogens, but further characterization of TOPs and their peptide substrates is required to understand their contributions to stress perception and defense signaling. Herein, label-free peptidomics via liquid chromatography-tandem mass spectrometry was used to differentially quantify 1111 peptides, originating from 369 proteins, between the Arabidopsis Col-0 wild type and top1top2 knock-out mutant. This revealed 350 peptides as significantly more abundant in the mutant, representing accumulation of these potential TOP substrates. Ten direct substrates were validated using in vitro enzyme assays with recombinant TOPs and synthetic candidate peptides. These TOP substrates are derived from proteins involved in photosynthesis, glycolysis, protein folding, biogenesis, and antioxidant defense, implicating TOP involvement in processes aside from defense signaling. Sequence motif analysis revealed TOP cleavage preference for non-polar residues in the positions surrounding the cleavage site. Identification of these substrates provides a framework for TOP signaling networks, through which the interplay between proteolytic pathways and defense signaling can be further characterized.     

Interaction of PHM, PHI and 24-glutamine PHI with human VIP receptors from colonic epithelium: comparison with rat intestinal receptors

PHM, the human counterpart of porcine Peptide Histidine Isoleucine amide (PHI), is shown to be a VIP agonist with low potency on human VIP receptors located in colonic epithelial cell membranes. Its potency is identical to that of PHI but by 3 orders of magnitude lower than that of VIP itself in inhibiting 125I-VIP binding and in stimulating adenylate cyclase activity. This contrasts markedly with the behaviour of PHI on rat VIP receptors located in intestinal epithelial cell membranes where PHI is a potent agonist with a potency that is 1/5 that of VIP. In another connection, we show that 24-glutamine PHI has the same affinity as 24-glutamic acid PHI (the natural peptide) for rat or human VIP receptors. These results indicate that while PHI may exert some physiological function through its interaction with VIP receptors in rodents, its human counterpart PHM is a very poor agonist of VIP in human. Furthermore, they show that the drastic change in position 24 of PHI (neutral versus acid residue) does not affect the activity of PHI, at least on VIP receptors.     

High-performance liquid chromatography and studies of neurophysin—neurohypophysial hormone pathways

High-performance liquid chromatography (HPLC) is being used extensively to characterize active polypeptides, precursor processing mechanisms, and cooperative peptide—protein noncovalent complexes in neuroendocrine pathways for neurohypophysial peptide hormones, oxytocin and vasopressin, and the hormone-associated proteins, neurophysins. Reversed-phase and ion-exchange HPLC polypeptide mapping have been used to detect the hormones, associated proteins, and other molecular forms containing these. This mapping but also ultimately to identify anatomical sites which contain the neurophysin/ hormone molecular pathways and to define the relatedness of polypeptide forms contained in different pathways. Reversed-phase HPLC also has provided a means to study proteolytic precursor processing, both to isolate synthetic and semisynthetic polypeptides and intermediates produced by these reactions. Finally, bioaffinity HPLC is being evaluated as a separatory and analytical tool. The latter includes its use to characterize the noncovalent peptide—protein and protein—protein interactions which occur among the molecular forms of the neurophysin/hormone pathways. These experiments typify the impact of HPLC for both analytical and preparative separations in studies of biologically active peptides and proteins.     

Secretin, VIP, and PHI stimulate rat proximal duodenal surface epithelial bicarbonate secretion in vivo

The surface epithelial cells of the stomach and duodenum secrete bicarbonate at rest and in response to a number of agonists including the gastrointestinal hormones, glucagon, and GIP. Since those hormones with structural homology may have similar effects, the purpose of the present study was to examine the effect of graded doses (6, 24, and 96 nmol/kg) of pure porcine secretin, VIP, and PHI on bicarbonate secretion by the proximal duodenum containing Brunner's glands. Experiments were performed in vivo on unanesthetized Sprague-Dawley rats with chronic Thiry-Vella type loops of the proximal 2 cm of duodenum. The order of testing was random and only one hormone was tested on a single day. Compared to the saline control, each dose of VIP produced a significant increase in duodenal bicarbonate secretion in a dose-response manner. The two higher doses of secretin and only the 96 nmol/kg dose of PHI significantly increased bicarbonate output. The responses to 96 nmol/kg dose of secretin and VIP were similar, and each was significantly greater than observed with PHI. It is concluded that secretin and VIP stimulate proximal duodenal bicarbonate secretion and are more potent than PHI.