Functional and anatomical analyses of active spinal circuits in a mouse model of chronic pain.

ABSTRACT: Decades of efforts in elucidating pain mechanisms, including pharmacological, neuroanatomical, and physiological studies have provided insights into how nociceptive information transmits from the periphery to the brain and the locations receiving nociceptive signals. However, little is known about which specific stimulus-dependent activated neurons, amongst heterogeneous neural environments, discriminatively evoke the cognate pain behavior. We here shed light on the population of neurons in the spinal cord activated by a painful stimulus to identify chronic pain-dependent activated neuronal subsets using Fos2A-iCreER (TRAP2) mice. We have found a large number of neurons activated by a normally nonpainful stimulus in the spinal cord of spinal nerve-ligated mice, compared with sham. Neuronal activation was observed in laminae I and II outer under heat hyperalgesia. A large number of neurons in laminae II inner were activated in both mechanical allodynia and heat hyperalgesia conditions, while mechanical allodynia tends to be the only stimulus that activates cells at lamina II inner dorsal region. Neuroanatomical analyses using spinal cell markers identified a large number of spinal inhibitory neurons that are recruited by both mechanical allodynia and heat hyperalgesia. Of interest, spinal neurons expressing calretinin, calbindin, and parvalbumin were activated differently with distinct pain modalities (ie, mechanical allodynia vs heat hyperalgesia). Chemogenetic inhibition of those activated neurons significantly and specifically reduced the response to the pain stimulus associated with the stimulus modality originally given to the animals. These findings support the idea that spinal neuronal ensembles underlying nociceptive transmission undergo dynamic changes to regulate selective pain responses.

Oxytocin neurons in the paraventricular nucleus of the hypothalamus circuit-dependently regulates social behavior, which malfunctions in BTBR mouse model of autism.

Oxytocin (OXT) a neuropeptide synthesized in the hypothalamic nuclei has a variety of function including socio-emotional processes in mammals. While the neural circuits and signaling pathways in central OXT converge in the paraventricular nucleus of the hypothalamus (PVN), we illuminate specific function of discrete PVN OXT circuits, which connect to the medial amygdala (MeA) and the bed nucleus of the stria terminalis (BnST) in mouse models. The OXTPVN→BnST projections are innervated from entire portions of the PVN, while those OXTPVN→MeA projections are asymmetrically innervated from the posterior portion of the PVN. Compared with OXT neurons in B6 wild type mice, BTBR mice that are recognized as a behavior-based autism model exhibited defect in the OXTPVN→BnST projection. We demonstrate that chemogenetic activation of OXTPVN→MeA circuit enhances anxiety-like behavior and facilitates social approach behavior, while activation of OXTPVN→BnST circuit suppresses anxiety-like behavior along with inhibiting social approach. This chemogenetic manipulation on the OXTPVN→BnST circuit proves ineffective in BTBR mice. Accordingly, chemogenetic activation of OXTPVN neurons that stimulate both OXT circuits induces OXT receptor expressions in both MeA and BnST as with those by social encounter in B6 mice. The induction of OXT receptor genes in the BnST was not observed in BTBR mice. These data support the hypothesis that OXT circuits serve as a regulator for OXT signaling in PVN to control socio-emotional approach/avoidance behavior, and a defect of OXTPVN→BnST circuit contributes to autism-like social phenotypes in BTBR mice.

The NOP Receptor System in Neurological and Psychiatric Disorders: Discrepancies, Peculiarities and Clinical Progress in Developing Targeted Therapies.

The nociceptin opioid peptide (NOP) receptor and its endogenous ligand nociceptin/orphanin FQ (N/OFQ) are the fourth members of the opioid receptor and opioid peptide families. Although they have considerable sequence homology to the other family members, they are not considered opioid per se because they do not have pharmacological profiles similar to the other family members. The number of NOP receptors in the brain is higher than the other family members, and NOP receptors can be found throughout the brain. Because of the widespread distribution of NOP receptors, N/OFQ and other peptide and small molecule agonists and antagonists have extensive CNS activities. Originally thought to be anti-opioid, NOP receptor agonists block some opioid activities, potentiate others, and modulate other activities not affected by traditional opiates. Because the effect of receptor activation can be dependent upon site of administration, state of the animal, and other variables, the study of NOP receptors has been fraught with contradictions and inconsistencies. In this article, the actions and controversies pertaining to NOP receptor activation and inhibition are discussed with respect to CNS disorders including pain (acute, chronic, and migraine), drug abuse, anxiety and depression. In addition, progress towards clinical use of NOP receptor-directed compounds is discussed.

Chemogenetics drives paradigm change in the investigation of behavioral circuits and neural mechanisms underlying drug action.

Recent developments in chemogenetic approaches to the investigation of brain function have ushered in a paradigm change in the strategy for drug and behavior research and clinical drug-based medications. As the nature of the drug action is based on humoral regulation, it is a challenge to identify the neuronal mechanisms responsible for the expression of certain targeted behavior induced by drug application. The development of chemogenetic approaches has allowed researchers to control neural activities in targeted neurons through a toolbox, including engineered G protein-coupled receptors or ligand-gated ion channels together with exogenously inert synthetic ligands. This review provides a brief overview of the chemogenetics toolbox with an emphasis on the DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) technique used in rodent models, which is applicable to the investigation of how specific neural circuits regulate behavioral processes. The use of chemogenetics has had a significant impact on basic neuroscience for a better understanding of the relationships between brain activity and the expression of behaviors with cell- and circuit-specific orders. Furthermore, chemogenetics is potentially a useful tool to deconstruct the neuropathological mechanisms of mental diseases and its regulation by drug, and provide us with transformative therapeutics with medication. We also review recent findings in the use of chemogenetic techniques to uncover functional circuit connections of serotonergic neurons in rodent models.

NOP receptor agonist attenuates nitroglycerin-induced migraine-like symptoms in mice.

Migraine is an extraordinarily prevalent and disabling headache disorder that affects one billion people worldwide. Throbbing pain is one of several migraine symptoms including sensitivity to light (photophobia), sometimes to sounds, smell and touch. The basic mechanisms underlying migraine remain inadequately understood, and current treatments (with triptans being the primary standard of care) are not well tolerated by some patients. NOP (Nociceptin OPioid) receptors, the fourth member of the opioid receptor family, are expressed in the brain and periphery with particularly high expression known to be in trigeminal ganglia (TG). The aim of our study was to further explore the involvement of the NOP receptor system in migraine. To this end, we used immunohistochemistry to examine NOP receptor distribution in TG and trigeminal nucleus caudalus (TNC) in mice, including colocalization with specific cellular markers, and used nitroglycerin (NTG) models of migraine to assess the influence of the selective NOP receptor agonist, Ro 64-6198, on NTG-induced pain (sensitivity of paw and head using von Frey filaments) and photophobia in mice. Our immunohistochemical studies with NOP-eGFP knock-in mice indicate that NOP receptors are on the majority of neurons in the TG and are also very highly expressed in the TNC. In addition, Ro 64-6198 can dose dependently block NTG-induced paw and head allodynia, an effect that is blocked by the NOP antagonist, SB-612111. Moreover, Ro 64-6198, can decrease NTG-induced light sensitivity in mice. These results suggest that NOP receptor agonists should be futher explored as treatment for migraine symptoms. This article is part of the special issue on Neuropeptides.

NOP-Related Mechanisms in Pain and Analgesia.

Since the discovery of the NOP receptor and N/OFQ as the endogenous ligand, evidence has appeared demonstrating the involvement of this receptor system in pain. This was not surprising for members of the opioid receptor and peptide families, particularly since both the receptor and N/OFQ are highly expressed in brain regions involved in pain, spinal cord, and dorsal root ganglia. What has been surprising is the complicated picture that has emerged from 25 years of research. The original finding that N/OFQ decreased tail flick and hotplate latency, when administered i.c.v., led to the hypothesis that NOP receptor antagonists could have analgesic activity without abuse liability. However, as data accumulated, it became clear that not only the potency but the activity per se was different when N/OFQ or small molecule NOP agonists were administered in the brain versus the spinal cord and it also depended upon the pain assay used. When administered systemically, NOP receptor agonists are generally ineffective in attenuating heat pain but are antinociceptive in an acute inflammatory pain model. Most antagonists administered systemically have no antinociceptive activity of their own, even though selective peptide NOP antagonists have potent antinociceptive activity when administered i.c.v. Chronic pain models provide different results as well, as small molecule NOP receptor agonists have potent anti-allodynic and anti-hyperalgesic activity after systemic administration. A considerable number of electrophysiological and anatomical experiments, in particular with NOP-eGFP mice, have been conducted in an attempt to explain the complicated profile resulting from NOP receptor modulation, to examine receptor plasticity, and to elucidate mechanisms by which selective NOP agonists, bifunctional NOP/mu agonists, or NOP receptor antagonists modulate acute and chronic pain.

Influence of neuropathic pain on nicotinic acetylcholine receptor plasticity and behavioral responses to nicotine in rats.

Tobacco smoking is particularly evident in individuals experiencing chronic pain. This complex relationship is poorly understood at both molecular and behavioral levels. Here, we describe experiments aimed at understanding whether a chronic pain state induces neuroadaptations into the brain or peripheral nerves that involve nicotinic acetylcholine receptors (nAChRs) and whether these neuroadaptations directly lead to increased vulnerability to nicotine addiction or to the development of coping strategies to relieve pain symptoms. We found that ligation of the rat L5 spinal nerve led to a dramatic downregulation in the mRNA expression levels of all nAChR subunits examined in dorsal root ganglia and a time-dependent downregulation of discrete subunits, particularly in the cingulate cortex and the amygdala. Spinal nerve ligation and sham-operated rats showed minor or no changes in patterns of acquisition and motivation for nicotine taking. Spinal nerve ligation rats also showed similar vulnerability to nicotine seeking as sham animals when reinstatement was induced by nicotine-associated cues, but failed to reinstate lever pressing when relapse was induced by nicotine priming. Spinal nerve ligation and sham rats were equally sensitive to nicotine-induced anxiety-like behavior and antinociception; however, nicotine produced a potent and long-lasting antiallodynic effect in spinal nerve ligation rats. These results demonstrate that chronic pain leads to plasticity of nAChRs that do not directly facilitate nicotine addictive behaviors. Instead, nicotine potently decreases allodynia, an effect that could lead to increased nicotine consumption in chronic pain subjects.

Analysis of the distribution of spinal NOP receptors in a chronic pain model using NOP-eGFP knock-in mice.

BACKGROUND AND PURPOSE: The nociceptin/orphanin FQ opioid peptide (NOP) receptor system plays a significant role in the regulation of pain. This system functions differently in the spinal cord and brain. The mechanism by which the NOP receptor agonists regulate pain transmission in these regions is not clearly understood. Here, we investigate the peripheral and spinal NOP receptor distribution and antinociceptive effects of intrathecal nociceptin/orphanin FQ (N/OFQ) in chronic neuropathic pain. EXPERIMENTAL APPROACH: We used immunohistochemistry to determine changes in NOP receptor distribution triggered by spinal nerve ligation (SNL) using NOP-eGFP knock-in mice. Antinociceptive effects of intrathecal N/OFQ on SNL-mediated allodynia and heat/cold hyperalgesia were assessed in wild-type mice. KEY RESULTS: NOP-eGFP immunoreactivity was decreased by SNL in the spinal laminae I and II outer, regions that mediate noxious heat stimuli. In contrast, immunoreactivity of NOP-eGFP was unchanged in the ventral border of lamina II inner, which is an important region for the development of allodynia. NOP-eGFP expression was also decreased in a large number of primary afferents in the L4 dorsal root ganglion (DRG) of SNL mice. However, SNL mice showed increased sensitivity, compared to sham animals to the effects of i.t administered N/OFQ with respect to mechanical as well as thermal stimuli. CONCLUSIONS AND IMPLICATIONS: Our findings suggest that the spinal NOP receptor system attenuates injury-induced hyperalgesia by direct inhibition of the projection neurons in the spinal cord that send nociceptive signals to the brain and not by inhibiting presynaptic terminals of DRG neurons in the superficial lamina.

Highly Selective and Potent α4ß2 nAChR Antagonist Inhibits Nicotine Self-Administration and Reinstatement in Rats.

The α4ß2 nAChR is the most predominant subtype in the brain and is a well-known culprit for nicotine addiction. Previously we presented a series of α4ß2 nAChR selective compounds that were discovered from a mixture-based positional-scanning combinatorial library. Here we report further optimization identified highly potent and selective α4ß2 nAChR antagonists 5 (AP-202) and 13 (AP-211). Both compounds are devoid of in vitro agonist activity and are potent inhibitors of epibatidine-induced changes in membrane potential in cells containing α4ß2 nAChR, with IC50 values of approximately 10 nM, but are weak agonists in cells containing α3ß4 nAChR. In vivo studies show that 5 can significantly reduce operant nicotine self-administration and nicotine relapse-like behavior in rats at doses of 0.3 and 1 mg/kg. The pharmacokinetic data also indicate that 5, via sc administration, is rapidly absorbed into the blood, reaching maximal concentration within 10 min with a half-life of less than 1 h.

Identification of 5,6-dihydroimidazo[2,1-b]thiazoles as a new class of antimicrobial agents.

In an effort to develop novel antimicrobial agents against drug-resistant bacterial infections, 5,6-dihydroimidazo[2,1-b]thiazole compounds were synthesized and tested for their antimicrobial activity. Eight compounds comprised by two sub-scaffolds were identified as hits against methicillin-resistant Staphylococcus aureus (MRSA). These hits were modified at 6-position by replacing (S)-6 to (R)-6 configuration and the (R)-isomers increased their antimicrobial activities by two-fold. The most active compound showed a MIC90 value of 3.7µg/mL against MRSA in a standard microdilution bacterial growth inhibitory assay. This compound protected wax moth worms against MRSA at a dose of 5× MIC using a worm infectious model. This compound also exhibited inhibition of DNA gyrase activity in a DNA gyrase supercoil assay, suggesting the 5,6-dihydroimidazo[2,1-b]thiazoles may target DNA gyrase for the antimicrobial action.

Knock-In Mice with NOP-eGFP Receptors Identify Receptor Cellular and Regional Localization.

The nociceptin/orphanin FQ (NOP) receptor, the fourth member of the opioid receptor family, is involved in many processes common to the opioid receptors including pain and drug abuse. To better characterize receptor location and trafficking, knock-in mice were created by inserting the gene encoding enhanced green fluorescent protein (eGFP) into the NOP receptor gene (Oprl1) and producing mice expressing a functional NOP-eGFP C-terminal fusion in place of the native NOP receptor. The NOP-eGFP receptor was present in brain of homozygous knock-in animals in concentrations somewhat higher than in wild-type mice and was functional when tested for stimulation of [(35)S]GTPγS binding in vitro and in patch-clamp electrophysiology in dorsal root ganglia (DRG) neurons and hippocampal slices. Inhibition of morphine analgesia was equivalent when tested in knock-in and wild-type mice. Imaging revealed detailed neuroanatomy in brain, spinal cord, and DRG and was generally consistent with in vitro autoradiographic imaging of receptor location. Multicolor immunohistochemistry identified cells coexpressing various spinal cord and DRG cellular markers, as well as coexpression with µ-opioid receptors in DRG and brain regions. Both in tissue slices and primary cultures, the NOP-eGFP receptors appear throughout the cell body and in processes. These knock-in mice have NOP receptors that function both in vitro and in vivo and appear to be an exceptional tool to study receptor neuroanatomy and correlate with NOP receptor function. SIGNIFICANCE STATEMENT: The NOP receptor, the fourth member of the opioid receptor family, is involved in pain, drug abuse, and a number of other CNS processes. The regional and cellular distribution has been difficult to determine due to lack of validated antibodies for immunohistochemical analysis. To provide a new tool for the investigation of receptor localization, we have produced knock-in mice with a fluorescent-tagged NOP receptor in place of the native NOP receptor. These knock-in mice have NOP receptors that function both in vitro and in vivo and have provided a detailed characterization of NOP receptors in brain, spinal cord, and DRG neurons. They appear to be an exceptional tool to study receptor neuroanatomy and correlate with NOP receptor function.

Genome-wide biochemical analysis of Arabidopsis protein phosphatase using a wheat cell-free system.

Plant genome possesses over 100 protein phosphatase (PPase) genes that are key regulators of signal transduction via phosphorylation/dephosphorylation event. Here we report a comprehensive functional analysis of protein serine/threonine, dual-specificity and tyrosine phosphatases using recombinant PPases produced by wheat cell-free protein synthesis system. Eighty-two recombinant PPases were successfully produced using Arabidopsis full-length cDNA as templates. In vitro PPase assay was performed using phosphorylated myelin basic protein as substrate. Among the AtPPases examined, 26 serine/threonine, three dual-specificity and one tyrosine PPases exhibited catalytic activity, including 20 serine/threonine and one dual-specificity PPases that showed in vitro activities for the first time. Our study demonstrates genome-wide biochemical analysis of AtPPases using wheat cell-free system, and provides new information and insights on enzyme activities.

Peptides derived from the prohormone proNPQ/spexin are potent central modulators of cardiovascular and renal function and nociception.

Computational methods have led two groups to predict the endogenous presence of a highly conserved, amidated, 14-aa neuropeptide called either spexin or NPQ. NPQ/spexin is part of a larger prohormone that contains 3 sets of RR residues, suggesting that it could yield more than one bioactive peptide; however, no in vivo activity has been demonstrated for any peptide processed from this precursor. Here we demonstrate biological activity for two peptides present within proNPQ/spexin. NPQ/spexin (NWTPQAMLYLKGAQ-NH(2)) and NPQ 53-70 (FISDQSRRKDLSDRPLPE) have differing renal and cardiovascular effects when administered intracerebroventricularly or intravenously into rats. Intracerebroventricular injection of NPQ/spexin produced a 13 ± 2 mmHg increase in mean arterial pressure, a 38 ± 8 bpm decrease in heart rate, and a profound decrease in urine flow rate. Intracerebroventricular administration of NPQ 53-70 produced a 26 ± 9 bpm decrease in heart rate with no change in mean arterial pressure, and a marked increase in urine flow rate. Intraventricular NPQ/spexin and NPQ 53-70 also produced antinociceptive activity in the warm water tail withdrawal assay in mice (ED(50)<30 and 10 nmol for NPQ/spexin and NPQ 53-70, respectively). We conclude that newly identified peptides derived from the NPQ/spexin precursor contribute to CNS-mediated control of arterial blood pressure and salt and water balance and modulate nociceptive responses.

Dynamic modulation of prohormone convertase 2 (PC2)-mediated precursor processing by 7B2 protein: preferential effect on glucagon synthesis.

The small neuroendocrine protein 7B2 is required for the production of active prohormone convertase 2 (PC2), an enzyme involved in the synthesis of peptide hormones, such as glucagon and proopiomelanocortin-derived α-melanocyte-stimulating hormone. However, whether 7B2 can dynamically modulate peptide production through regulation of PC2 activity remains unclear. Infection of the pancreatic alpha cell line α-TC6 with 7B2-encoding adenovirus efficiently increased production of glucagon, whereas siRNA-mediated knockdown of 7B2 significantly decreased stored glucagon. Furthermore, rescue of 7B2 expression in primary pituitary cultures prepared from 7B2 null mice restored melanocyte-stimulating hormone production, substantiating the role of 7B2 as a regulatory factor in peptide biosynthesis. In anterior pituitary and pancreatic beta cell lines, however, overexpression of 7B2 affected neither production nor secretion of peptides despite increased release of active PC2. In direct contrast, 7B2 overexpression decreased the secretion and increased the activity of PC2 within α-TC6 cells; the increased intracellular concentration of active PC2 within these cells may therefore account for the enhanced production of glucagon. In line with these findings, we found elevated circulating glucagon levels in 7B2-overexpressing cast/cast mice in vivo. Surprisingly, when proopiomelanocortin and proglucagon were co-expressed in either pituitary or pancreatic alpha cell lines, proglucagon processing was preferentially decreased when 7B2 was knocked down. Taken together, these results suggest that proglucagon cleavage has a greater dependence on PC2 activity than other precursors and moreover that 7B2-dependent routing of PC2 to secretory granules is cell line-specific. The manipulation of 7B2 could therefore represent an effective way to selectively regulate synthesis of certain PC2-dependent peptides.

Processing of proaugurin is required to suppress proliferation of tumor cell lines.

Augurin is a secretory molecule produced in pituitary, thyroid, and esophagus and implicated in a wide array of physiological processes, from ACTH release to tumor suppression. However, the specific proaugurin-derived peptides present in various cell types are not yet known. In order to shed light on the posttranslational modifications required for biological activity, we here describe the posttranslational processing of proaugurin in AtT-20 and Lovo cells and identify proaugurin-derived products generated by convertases. In vitro cleavage of proaugurin with proprotein convertases produced multiple peptides, including a major product with a mass of 9.7 kDa by mass spectrometry. Metabolic labeling of C-terminally tagged proaugurin in AtT-20 and AtT-20/PC2 cells resulted in a major 15-kDa tagged form on SDS-PAGE, which likely corresponds to the 9.7-kDa in vitro fragment, with the added tag, its linker, and posttranslational modification(s). The secretion of neither proaugurin nor this cleavage product was stimulated by forskolin, indicating its lack of storage in regulated secretory granules and lack of cleavage by PC2. Incubation of cells with the furin inhibitor nona-d-arginine resulted in impaired cleavage of proaugurin, whereas metalloprotease inhibitors did not affect proaugurin proteolysis. These data support the idea that proaugurin is cleaved by furin and secreted via the constitutive secretory pathway. Interestingly, proaugurin was sulfated during trafficking; sulfation was completely inhibited by brefeldin A. Proliferation assays with three different tumor cell lines demonstrated that only furin-cleaved proaugurin could suppress cell proliferation, suggesting that proteolytic cleavage is a posttranslational requirement for proaugurin to suppress cell proliferation.

Modulation of prohormone convertase 1/3 properties using site-directed mutagenesis.

Prohormone convertase (PC)1/3 and PC2 cleave active peptide hormones and neuropeptides from precursor proteins. Compared with PC2, recombinant PC1/3 exhibits a very low specific activity against both small fluorogenic peptides and recombinant precursors, even though the catalytic domains in mouse PC1/3 and PC2 share 56% amino acid sequence identity. In this report, we have designed PC2-specific mutations into the catalytic domain of PC1/3 in order to investigate the molecular contributions of these sequences to PC1/3-specific properties. The exchange of residues RQG(314) with the SY sequence present in the same location within PC2 paradoxically shifted the pH optimum of PC1/3 upward into the neutral range; other mutations in the catalytic domain had no effect. Although none of the full-length PC1/3 mutants examined exhibited increased specific activity, the 66-kDa form of the RQG(314)SY mutant was two to four times more active than the 66-kDa form of wild-type PC1/3. However, stable transfection of RQG(314)SY into PC12 cells did not result in greater activity against the endogenous substrate proneurotensin, implying unknown cellular controls of PC1/3 activity. Mutation of GIVTDA(243-248) to QPFMTDI, a molecular determinant of 7B2 binding, resulted in increased zymogen expression but no propeptide cleavage or secretion, suggesting that this mutant is trapped in the endoplasmic reticulum due to an inability to cleave its own propeptide. We conclude that many convertase-specific properties are attributable less to convertase-specific catalytic cleft residues than to convertase-specific domain interactions.

Deorphanization of novel peptides and their receptors.

Peptide hormones and neuropeptides play important roles in endocrine and neural signaling, often using G protein-coupled receptor (GPCR)-mediated signaling pathways. However, the rate of novel peptide discovery has slowed dramatically in recent years. Genomic sequencing efforts have yielded a large number of cDNA sequences that potentially encode novel candidate peptide precursors, as well as hundreds of orphan GPCRs with no known cognate ligands. The complexity of peptide signaling is further highlighted by the requirement for specific posttranslational processing steps, and these must be accomplished in vitro prior to testing newly discovered peptide precursor candidates in receptor assays. In this review, we present historic as well as current approaches to peptide discovery and GPCR deorphanization. We conclude that parallel and combinatorial discovery methods are likely to represent the most fruitful avenues for both peptide discovery as well as for matching the remaining GPCRs with their peptide ligands.

Enzymatic characterization of a human acyltransferase activity.

BACKGROUND: Non-histone protein acylation is increasingly recognized as an important posttranslational modification, but little is known as to the biochemical properties of protein serine acylating enzymes. METHODOLOGY/PRINCIPAL FINDINGS: We here report that we have identified a metal-stimulated serine octanoyltransferase activity in microsomes from human erythroleukemic (HEL) cells. The HEL acylating enzyme was linear with respect to time and protein, exhibited a neutral pH optimum (stimulated by cobalt and zinc), and inhibited by chelating reagents. Hydroxylamine treatment removed most, but not all, of the attached radioactivity. A salt extract of microsomal membranes contained the major portion of enzyme activity, indicating that this acyltransferase is not an integral membrane protein. Sucrose density fractionation showed that the acyltransferase activity is concentrated in the endoplasmic reticulum. In competition experiments, the acyltransferase was well inhibited by activated forms of fatty acids containing at least eight to fourteen carbons, but not by acetyl CoA. The zinc-stimulated HEL acyltransferase did not octanoylate proenkephalin, proopiomelanocortin, His-tagged proghrelin, or proghrelin lacking the amino-terminal His-tag stub of Gly-Ala-Met. The peptides des-acyl ghrelin and ACTH were also not acylated; however, des-acyl ghrelin containing the N-terminal tripeptide Gly-Ala-Met was acylated. Mutagenesis studies indicated a requirement for serine five residues from the amino terminus, reminiscent of myristoyl transferase, but not of ghrelin acylation. However, recombinant myristoyl transferase could not recapitulate the hydroxylamine sensitivity, zinc-stimulation, nor EDTA inhibition obtained with HEL acyltransferase, properties preserved in the HEL cell enzyme purified through four sequential chromatographic steps. CONCLUSIONS/SIGNIFICANCE: In conclusion, our data demonstrate the presence of a zinc-stimulated acyltransferase activity concentrated in the endoplasmic reticulum in HEL cells which is likely to contribute to medium-chain protein lipidation.

Synthetic small-molecule prohormone convertase 2 inhibitors.

The proprotein convertases are believed to be responsible for the proteolytic maturation of a large number of peptide hormone precursors. Although potent furin inhibitors have been identified, thus far, no small-molecule prohormone convertase 1/3 or prohormone convertase 2 (PC2) inhibitors have been described. After screening 38 small-molecule positional scanning libraries against recombinant mouse PC2, two promising chemical scaffolds were identified: bicyclic guanidines, and pyrrolidine bis-piperazines. A set of individual compounds was designed from each library and tested against PC2. Pyrrolidine bis-piperazines were irreversible, time-dependent inhibitors of PC2, exhibiting noncompetitive inhibition kinetics; the most potent inhibitor exhibited a K(i) value for PC2 of 0.54 microM. In contrast, the most potent bicyclic guanidine inhibitor exhibited a K(i) value of 3.3 microM. Cross-reactivity with other convertases was limited: pyrrolidine bis-piperazines exhibited K(i) values greater than 25 microM for PC1/3 or furin, whereas the K(i) values of bicyclic guanidines for these other convertases were more than 15 microM. We conclude that pyrrolidine bis-piperazines and bicyclic guanidines represent promising initial leads for the optimization of therapeutically active PC2 inhibitors. PC2-specific inhibitors may be useful in the pharmacological blockade of PC2-dependent cleavage events, such as glucagon production in the pancreas and ectopic peptide production in small-cell carcinoma, and to study PC2-dependent proteolytic events, such as opioid peptide production.

Production of bioactive peptides in an in vitro system.

An in vitro system for the preparation of bioactive peptides is described. This system couples three different posttranslational modification enzymes, prohormone convertases (PCs), carboxypeptidase E, and peptidyl alpha-amidating enzyme, to transform recombinant precursors into bioactive peptides. Three different precursors, mouse proopiomelanocortin (mPOMC), rat proenkephalin (rPE), and human proghrelin, were used as model systems. The conversion of mPOMC and rPE to smaller peptide products was measured by radioimmunoassay. After optimization of the system, excellent efficiency was obtained: about 85% of starting mPOMC was converted to des-acetyl alpha-melanocyte-stimulating hormone (alpha-MSH). For proenkephalin, 75 and 96% yields were obtained for the opioid peptides Met-RGL and Met-enk, respectively. Cell-based assays demonstrated that in-vitro-generated des-acetyl alpha-MSH successfully activated the melanocortin 4 receptor. Proghrelin digestion was used to screen the specificity of PC cleavage and to confirm the cleavage site by mass spectroscopy. Mature ghrelin was produced by human furin, mouse prohormone convertase 1, and human prohormone convertase 7 but not by mouse prohormone convertase 2. These results demonstrate that our in vitro system (1) can produce peptides in quantities sufficient to carry out functional analyses, (2) can be used to determine the specificity of proprotein convertases on recombinant precursors, and (3) has the potential to identify novel peptide functions on both known and orphan G-protein-coupled receptors.