Robert Day

Gene expression of prohormone and proprotein convertases in the rat CNS: a comparative in situ hybridization analysis

Posttranslational processing of proproteins and prohormones is an essential step in the formation of bioactive peptides, which is of particular importance in the nervous system. Following a long search for the enzymes responsible for protein precursor cleavage, a family of Kexin/subtilisin-like convertases known as PC1, PC2, and furin have recently been characterized in mammalian species. Their presence in endocrine and neuroendocrine tissues has been demonstrated. This study examines the mRNA distribution of these convertases in the rat CNS and compares their expression with the previously characterized processing enzymes carboxypeptidase E (CPE) and peptidylglycine alpha-amidating monooxygenase (PAM) using in situ hybridization histochemistry. Furin mRNA was ubiquitously distributed and detected both in neurons and non- neuronal tissue throughout the brain with a higher abundance in ependyma, the circumventricular organs, the islands of Calleja, hippocampus, and allocortex. The cellular localization of PC1 and PC2 was exclusively neuronal with highest concentrations in known neuropeptide-rich brain regions. In general, PC2 was more widely expressed than PC1 in the CNS, although many regional variations were detected. The identification of specific combinations of convertase expression together with CPE and PAM expression in neuropeptide-rich brain regions suggests that specific enzymatic pathways are involved in neuropeptide precursor processing, and that these specific combinations are responsible for region-specific differences of posttranslational processing.

Chronic electroconvulsive shock treatment elicits up-regulation of CRF and AVP mRNA in select populations of neuroendocrine neurons

The effects of repeated electroconvulsive seizures (ECS) on expression of mRNAs coding for corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) in neuroendocrine neurons of the hypothalamo-pituitary-adrenocortical (HPA) axis and hypothalamo-neurohypophysial system (HNS) were assessed via semi-quantitative in situ hybridization histochemical analysis. Measures of mRNA content were accompanied by measurement of peptide- and hormone-expression in the relevant neuroendocrine systems. Following 7 daily ECS treatments, CRF mRNA was significantly increased in the medial parvocellular paraventricular nucleus (PVN) of treated rats relative to controls. CRF peptide content of whole PVN homogenates was decreased to 50% of control levels. Changes in CRF message and peptide levels were accompanied by increases in pituitary ACTH content and by elevated plasma corticosterone, suggesting ECS elicits long-term up-regulation of the HPA axis. AVP mRNA in the medial parvocellular PVN, which is known to up-regulate in response to HPA challenge by adrenalectomy, was not increased by ECS. Chronic ECS causes a clear up-regulation of HNS neurons of the supraoptic nucleus, characterized by increased AVP mRNA content, decreased AVP peptide content, and depletion of neurohypophysial AVP. However, no changes were observed in magnocellular vasopressinergic neurons of the PVN, indicating that magnocellular SON and PVN neurons respond differentially to stimulation by ECS. The data indicate that ECS is a potent stimulus for activation of select components of both the HPA axis and the HNS. As such, ECS provides a useful tool for examining mechanisms underlying neuroendocrine processes.

Regulation of striatonigral prodynorphin peptides by dopaminergic agents

The primary purpose of this study was to examine the regulation of prodynorphin peptides by dopaminergic agents in the central nervous system. The indirectly acting catecholamine agonist D-amphetamine sulfate (AMPH) and the dopamine receptor antagonist haloperidol (HAL) were administered to rats across a variety of treatment schedules and drug doses. The striatum, substantia nigra and hippocampus were dissected and examined by radioimmunoassay for 5 different prodynorphin peptides, covering all 3 opioid domains in the prodynorphin precursor: dynorphin A(1-8) and dynorphin A(1-17) of the dynorphin A domain, dynorphin B(1-13) of the dynorphin B domain, and alpha-neo-endorphin and beta-neo-endorphin of the neo-endorphin domain. In addition, the proenkephalin peptide Met-enkephalin-arg6-gly7-leu8 (MERGL) was examined in the striatum. AMPH administered one hour prior to sacrifice caused a dose-dependent depletion of prodynorphin peptides in both the striatum and substantia nigra. In animals treated with AMPH once each day for 7 days and sacrificed 24 h later, a dramatic dose-dependent increase in prodynorphin peptides was observed in these brain regions. Animals treated with AMPH once each day for 7 days and sacrificed one hour after the final injection showed no changes in prodynorphin peptides. In addition to changes in individual prodynorphin peptides, AMPH treatment caused alterations in the relationships between intermediate peptides (dynorphin A(1-17) and alpha-neo-endorphin) and their immediate products (dynorphin A(1-8) and beta-neo-endorphin). AMPH caused no consistent changes in prodynorphin peptides in the hippocampus, or in MERGL in the striatum. Taken together these data suggest that acute dopaminergic activation causes depletion of dynorphins from striatonigral prodynorphin neurons, presumably due to dopamine-dependent release of these peptides; repeated activation causes repeated release, with a rebound increase in biosynthesis. HAL, in contrast to AMPH caused relatively subtle changes in striatonigral prodynorphin peptides. Although no significant changes in individual prodynorphin peptides were observed, HAL treatment caused a change in the relationship between dynorphin A(1-17) and dynorphin A(1-8), a change opposite in direction to that observed with AMPH treatment. As has been previously reported, repeated HAL administration caused a dose-dependent increase in the proenkephalin peptide MERGL. The relatively subtle effects of HAL on prodynorphin peptides suggests that tonic dopamine activity is not important in the regulation of striatonigral prodynorphin neurons. The potential functional and behavioral significance of the present results are discussed.

Proenkephalin messenger RNA is expressed both in the rat anterior and posterior pituitary

The presence of proenkephalin (PENK)-derived opioid peptides in the pituitary gland is well known. However, the cellular sources of their biosynthetic origin in all three pituitary lobes are less clear. In this study we identified the potential sites of synthesis by localizing the mRNA coding for PENK in the rat pituitary gland using in situ hybridization histochemistry. Numerous cells containing PENK mRNA were detected throughout the anterior lobe. Although suggested by previous reports, no mRNA signal could be detected in the intermediate lobe. Surprisingly, high levels of PENK mRNA were found in the posterior lobe. The cellular distribution in the neural lobe implies that pituicytes, a special class of glial cells, may express PENK mRNA.

Neuroanatomical and functional studies of peptide precursor-processing enzymes

An overview of in situ hybridization mapping studies comparing the brain distributions of mRNA transcripts encoding the proprotein convertase Furin, PC1 and PC2 in relation to transcripts encoding carboxypeptidase H (CPE) and peptidylglycine alpha-amidating monooxygenase (PAM) is presented. Furin mRNA was detected in both neurons and non-neuronal cells throughout all brain areas. The cellular localization of PC1 and PC2 was primarily neuronal, with PC2 generally more widely distributed, although many regional variations were detected. The detection of specific combinations of the convertases, CPE and PAM in peptide-rich brain regions suggests that specific enzymatic pathways are involved in neuropeptide processing. Results are also described from a series of functional studies on the processing of pro-opiomelanocortin (POMC) in a heterologous neuronal cell line, Neuro-2A, which expresses low levels of PC2 mRNA but no detectable PC1 mRNA. Two contrasting POMC-processing patterns were observed: one where the precursor was processed at a number of cleavage sites to produce several peptides, and another where POMC was processed at a single cleavage site to produce beta E only. If PC2 is responsible for POMC processing in transfected cells, this enzyme may have favored cleavage of the amino terminal-processing site above other sites in the latter type of cell line.

Structure-Based Optimization of a Potent PACE4 Inhibitor Containing a Decarboxylated P1 Arginine Mimetic

Our recent studies have provided direct evidence for the critical role of PACE4 in the progression of prostrate cancer, identifying this enzyme as a promising target to design novel and effective treatments [1]. Moreover, we developed a potent PACE4 inhibitor with considerable selectivity (20-fold over furin) known as the Multi-Leu (ML) peptide [2]. In order to improve its pharmacological profile, we performed structure-activity relationship (SAR) studies and determined that the incorporation of the decarboxylated arginine mimetic (4-amidinobenzylamide, Amba) at the P1 position led to a more potent and stable analog [3]. Unfortunately, this inhibitor suffered from a reduced selectivity towards PACE4. To restore its specificity profile, we used a positional-scanning approach and synthesized peptide libraries by substituting each amino acid residue in the leucine core of our inhibitor. These studies revealed that we are able to enhance the specificity profile (3-fold) and preserve the inhibitory activity as well as antiproliferative properties of our inhibitor by incorporating a leucine isomer – L-isoleucine into its structure (Maluch, et al., unpublished data). Based on these results, we decided to perform further SAR studies aiming to improve the specificity and activity of our MLAmba inhibitor. We focused on the leucine core (P8-P5) and its modification with unnatural amino acid residues possessing hydrophobic character (Figure 1). First we evaluated the impact of a single substitution (from the P8 to P5 position) on the inhibitory activity of the resulting peptides, and then we combined the most promising modifications. In this work, we present the synthesis and biological evaluation of a new series of MLAmba analogs.

Structural Determinants of Furin Inhibitors Derived from Influenza Hemagglutinin

Furin belongs to a small family of the proprotein convertases (PCs) and is the best-characterized member with a ubiquitous tissue distribution. Furin is responsible for posttranslational transformation and activation of proproteins into biologically active proteins and regulation of many physiological processes in living organisms [1]. In addition to their normal physiological role, furin and related convertases contribute to the maturation of many diseases-related proteins and are involved in tumorigenesis, neurodegenerative disorders, diabetes and atherosclerosis [2]. Moreover, furin is also required for the activation of many bacterial and viral toxin precursors (including Pseudomonas aeruginosa, pathogenic Ebola strains, Marburg, HIV gp160, the avian influenza virus hemagglutinin) [3]. Studies have shown that the hemagglutinin of avian influenza virus A contains a TPRERRRKKRTGL sequence, which is a furin cleavage site. The optimization of TPRERRRKKRTGL peptide led to the discovery of a potent inhibitor with the following sequence: Ac-RARRRKKRT-NH2 [4,5]. In the present work, the structural determinants for furin inhibition were investigated using positional-scanning approach. We synthesized peptide libraries substituted by each natural amino acid residue (with the exception of the Cys) in the P5-P8 positions, while maintaining the furin recognition motif (the P4-P1 positions) and we determined inhibitor activity of the resulting peptides towards recombinant furin.