Biomedical subjects

Marc J Glucksman

Publications and source records attributed to Marc J Glucksman.

16 recordsLinked to original sources

PubMed2006

Modulation of bradykinin signaling by EP24.15 and EP24.16 in cultured trigeminal ganglia.

Metalloendopeptidases expressed in neural tissue are characterized in terms of their neuropeptide substrates. One such neuropeptide, bradykinin (BK), is an important inflammatory mediator that activates the type-2 BK receptor (B2R) on the terminal endings of specialized pain-sensing neurons known as nociceptors. Among several metalloendopeptidases that metabolize and inactivate BK, EP24.15 and EP24.16 are known to associate with the plasma membrane in several immortalized cell lines. Potentially, the colocalization of EP24.15/16 and B2R at plasma membrane microdomains known as lipid rafts in a physiologically relevant nociceptive system would allow for discrete, peptidase regulation of BK signaling. Western blot analysis of crude subcellular fractions and lipid raft preparations of cultured rat trigeminal ganglia demonstrate similar expression profiles between EP24.15/16 and B2R on a subcellular level. Furthermore, the treatment of primary cultures of trigeminal ganglia with inhibitors of EP24.15/16 led to the potentiation of several bradykinin-induced events that occur downstream of B2R activation. EP24.15/16 inhibition by N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-AlalTyr-p-aminobenzoate (cFP) resulted in a 1000-fold increase in B2R sensitivity to BK as measured by inositol phosphate accumulation. In addition, cFP treatment resulted in a 31.1+/-5.0% potentiation of the ability of BK to inhibit protein kinase B (Akt) activity. Taken together, these data demonstrate that EP24.15/16 modulate intracellular, peptidergic signaling cascades through B2R in a physiologically relevant nociceptive system.

Animals↗

PubMed2006

Facilitation of lordosis in rats by a metabolite of luteinizing hormone releasing hormone.

In the female rat, ovulation is preceded by a marked increase in the release of the decapeptide, LHRH, culminating in a preovulatory LH surge, which coincides with a period of sexual receptivity. The decapeptide, LHRH, is processed by a zinc metalloendopeptidase EC 3.4.24.15 (EP24.15) that cleaves the hormone at the Tyr(5)-Gly(6) bond. We have previously reported that the autoregulation of LHRH gene expression can also be mediated by its metabolite, LHRH-(1-5). Given the central function of LHRH in reproduction and reproductive behavior, we examined the role of the metabolite, LHRH-(1-5), in mediation of LHRH-facilitated reproductive behavior. Intracerebroventricular administration of LHRH-(1-5) facilitated sexual behavior responses, similar to those facilitated by the decapeptide LHRH, in ovariectomized estradiol-primed female rats. Furthermore, immunoneutralization of EP24.15 resulted in the inhibition of the LHRH-facilitated lordosis but had no inhibitory effects on LHRH-(1-5)-facilitated lordosis. The LHRH antagonist, Antide, was capable of inhibiting LHRH-facilitated lordosis, without affecting LHRH-(1-5)-facilitated lordosis. Collectively, these results suggest a role for LHRH metabolites in the facilitation of female receptive behavior in rats.

Analysis of Variance↗

PubMed2006

Mapping protease substrates by using a biotinylated phage substrate library.

We describe a bacteriophage M13 substrate library encoding the AviTag (BirA substrate) and combinatorial heptamer peptides displayed at the N terminus of the mature form of capsid protein III. Phages are biotinylated efficiently (> or = 50%) when grown in E. coli cells coexpressing BirA, and such viral particles can be immobilized on a streptavidin-coated support and released by protease cleavage within the combinatorial peptide. We have used this library to map the specificity of human Factor Xa and a neuropeptidase, neurolysin (EC3.4.24.16). Validation by analysis of isolated peptide substrates has revealed that neurolysin recognizes the motif hydrophobic-X-Pro-Arg-hydrophobic, where Arg-hydrophobic is the scissile bond.

Bacteriophage M13↗

PubMed2005

A prohormone convertase cleavage site within a predicted alpha-helix mediates sorting of the neuronal and endocrine polypeptide VGF into the regulated secretory pathway.

Distinct intracellular pathways are involved in regulated and constitutive protein secretion from neuronal and endocrine cells, yet the peptide signals and molecular mechanisms responsible for targeting and retention of soluble proteins in secretory granules are incompletely understood. By using confocal microscopy and subcellular fractionation, we examined trafficking of the neuronal and endocrine peptide precursor VGF that is stored in large dense core vesicles and undergoes regulated secretion. VGF cofractionated with secretory vesicle membranes but was not detected in detergent-resistant lipid rafts. Deletional analysis using epitope-tagged VGF suggested that the C-terminal 73-amino acid fragment of VGF, containing two predicted alpha-helical loops and four potential prohormone convertase (PC) cleavage sites, was necessary and sufficient with an N-terminal signal peptide-containing domain, for large dense core vesicle sorting and regulated secretion from PC12 and INS-1 cells. Further transfection analysis identified the sorting sequence as a compact C-terminal alpha-helix and embedded 564RRR566 PC cleavage site; mutation of the 564RRR566 PC site in VGF-(1-65): GFP:VGF-(545-617) blocked regulated secretion, whereas disruption of the alpha-helix had no effect. Mutation of the adjacent 567HFHH570 motif, a charged region that might enhance PC cleavage in acidic environments, also blocked regulated release. Finally, inhibition of PC cleavage in PC12 cells using the membrane-permeable synthetic peptide chloromethyl ketone (decanoyl-RVKR-CMK) blocked regulated secretion of VGF. Our studies define a critical RRR-containing C-terminal domain that targets VGF into the regulated pathway in neuronal PC12 and endocrine INS-1 cells, providing additional support for the proposed role that PCs and their cleavage sites play in regulated peptide secretion.

Amino Acid Motifs↗

PubMed2005

Flexibility in substrate recognition by thimet oligopeptidase as revealed by denaturation studies.

Thimet oligopeptidase (TOP) is a soluble metalloendopeptidase belonging to a family of enzymes including neurolysin and neprilysin that utilize the HEXXH metal-binding motif. TOP is widely distributed among cell types and is able to cleave a number of structurally unrelated peptides. A recent focus of interest has been on structure-function relationships in substrate selectivity by TOP. The enzyme's structural fold comprises two domains that are linked at the bottom of a deep substrate-binding cleft via several flexible loop structures. In the present study, fluorescence spectroscopy has been used to probe structural changes in TOP induced by the chemical denaturant urea. Fluorescence emission, anisotropy and collisional quenching data support a two-step unfolding process for the enzyme in which complete loss of the tertiary structure occurs in the second step. Complete loss of activity and loss of catalytic Zn(II) from the active site, monitored by absorption changes of the metal chelator 4-(2-pyridylazo)-resorcinol, are also connected with the second step. In contrast, the first unfolding event, which is linked to changes in the non-catalytic domain, leads to a sharp increase in kcat towards a 9-residue substrate and a sharp decrease in kcat for a 5-residue substrate. Thus a conformational change in TOP has been directly correlated with a change in substrate selectivity. These results provide insight into how the enzyme can process the range of structurally unrelated peptides necessary for its many physiological roles.

Enzyme Stability↗

PubMed2005

Calcium modulates endopeptidase 24.15 (EC 3.4.24.15) membrane association, secondary structure and substrate specificity.

The metalloendopeptidase 24.15 (EP24.15) is ubiquitously present in the extracellular environment as a secreted protein. Outside the cell, this enzyme degrades several neuropeptides containing from 5 to 17 amino acids (e.g. gonadotropin releasing hormone, bradykinin, opioids and neurotensin). The constitutive secretion of EP24.15 from glioma C6 cells was demonstrated to be stimulated linearly by reduced concentrations of extracellular calcium. In the present report we demonstrate that extracellular calcium concentration has no effect on the total amount of the extracellular (cell associated + medium) enzyme. Indeed, immuno-cytochemical analyses by confocal and electron microscopy suggested that the absence of calcium favors the enzyme shedding from the plasma membrane into the medium. Two putative calcium-binding sites on EP24.15 (D93 and D159) were altered by site-directed mutagenesis to investigate their possible contribution to binding of the enzyme at the cell surface. These mutated recombinant proteins behave similarly to the wild-type enzyme regarding enzymatic activity, secondary structure, calcium sensitivity and immunoreactivity. However, immunocytochemical analyses by confocal microscopy consistently show a reduced ability of the D93A mutant to associate with the plasma membrane of glioma C6 cells when compared with the wild-type enzyme. These data and the model of the enzyme's structure as determined by X-ray diffraction suggest that D93 is located at the enzyme surface and is consistent with membrane association of EP24.15. Moreover, calcium was also observed to induce a major change in the EP24.15 cleavage site on distinctive fluorogenic substrates. These data suggest that calcium may be an important modulator of ep24.15 cell function.

Animals↗

PubMed2004

Stimulation of luteinizing hormone-releasing hormone (LHRH) gene expression in GT1-7 cells by its metabolite, LHRH-(1-5).

Given the central role of the decapeptide LHRH in reproduction and reproductive behavior, it is important to focus on delineating the possible effects of this gene and its products in the regulation of hormone-dependent reproductive processes. In the female, ovulation is preceded by a marked increase in LHRH release; the increase in LHRH release culminates in a preovulatory LH surge, which coincides with a period of sexual receptivity. In contrast to the belief that the proteolytic metabolism of LHRH serves only as a degradative process that removes excess LHRH and attenuates signal transduction through the LHRH receptor, we hypothesized that a metabolite of the decapeptide, LHRH-(1-5), can directly regulate LHRH neuronal function. This study demonstrates the ability of LHRH-(1-5) peptide to regulate LHRH gene expression in the LHRH neuronal cell line, the GT(1-7) cell. The results show that LHRH-(1-5) stimulated LHRH gene expression at the posttranscriptional level. In contrast to the LHRH suppression of its own gene expression, the coadministration of LHRH with the metalloendopeptidase, EC 3.4.24.15, an endopeptidase known to cleave LHRH to form LHRH(1-5), shows a reversal of effect, a stimulation of LHRH gene expression. Finally, the effect of LHRH-(1-5) on LHRH gene expression appears to be mediated by the calcium/calmodulin-dependent protein kinase. The present study supports the hypothesis that the physiological metabolite of LHRH, LHRH-(1-5), is functionally capable of regulating the reproductive neuroendocrine system.

Animals↗

PubMed2004

Regulation of endopeptidases EC3.4.24.15 and EC3.4.24.16 in vascular endothelial cells by cyclic strain: role of Gi protein signaling.

OBJECTIVE: Endopeptidase EC3.4.24.15 (EP24.15)- and EC3.4.24.16 (EP24.16)-specific peptide hydrolysis plays an important role in endothelium-mediated vasoregulation. Given the significant influence of hemodynamic forces on vascular homeostasis and pathology, we postulated that these related peptidases may be mechanosensitive. The objective of this study, therefore, was to investigate the putative role of cyclic strain in regulating the expression and enzymatic activity of EP24.15 and EP24.16 in bovine aortic endothelial cells (BAECs). METHODS AND RESULTS: BAECs were cultured under conditions of defined cyclic strain (0% to 10% stretch, 60 cycles/min, 0 to 24 hours). Strain significantly increased EP24.15 and EP24.16 soluble activity in a force- and time-dependent manner, with elevations of 2.3+/-0.4- and 1.9+/-0.3-fold for EP24.15 and EP24.16, respectively, after 24 hours at 10% strain. Pharmacological agents and dominant-negative G protein mutants used to selectively disrupt Gi(alpha)- and Gbetagamma-mediated signaling pathways attenuated strain-dependent (24 hours, 5%) increases for both enzymes. Differences in the inhibitory profile for both enzymes were also noted, with EP24.15 displaying greater sensitivity to Gi(alpha2/3) inhibition and EP24.16 exhibiting greater sensitivity to Gi(alpha1/2) and Gbetagamma inhibition. Cyclic strain also increased levels of secreted EP24.15 and EP24.16 activity by 2.6+/-0.02- and 3.6+/-0.2-fold, respectively, in addition to mRNA levels for both enzymes (EP24.15 +42%, EP24.16 +56%). CONCLUSIONS: Our findings suggest that cyclic strain putatively regulates both the mRNA expression and enzymatic function of EP24.15 and EP24.16 in BAECs via alternate Gi protein signaling pathways.

Animals↗

PubMed2004

Neuroproteomics: expression profiling of the brain's proteomes in health and disease.

The term "proteome" describes the protein complement of a genome. Proteomes of cells are dynamic and are directly affected by environmental factors, such as stress and/or drug treatment, or as a result of aging and disease. One of the distinct advantages of proteomic analysis, not attainable with RNA expression data, is the ability to fractionate the cell's proteins into various subpopulations. In neuroscience, "neuromics" (proteomics in the central nervous system) is in its infancy, with a paucity of studies in the context of the brain. One of the objectives of this review is to illustrate the potential of neuromics to profile differences in the distribution of thousands of proteins as a function of disease markers. We have previously used this approach to determine the effects of varied postmortem interval in examining human brain tissue and to identify biomarkers. Here we review proteomic studies of schizophrenia, Alzheimer's disease, and Parkinson's disease. Experimental results regarding Parkinson's disease are presented to illustrate the potential of neuromics to identify pathways of pathogenesis and novel therapeutic targets.

Alzheimer Disease↗

PubMed2004

Metalloendopeptidase EC3.4.24.15 is constitutively released from the exofacial leaflet of lipid rafts in GT1-7 cells.

Metalloendopeptidase EC3.4.24.15 (EP24.15) is a physiologically important neuropeptide-degrading enzyme involved in the metabolism of multiple neuropeptides. The mechanism of release of EP24.15 from neuronal cells is multimodal, being both constitutive and stimulatable. Previous studies have characterized stimulated EP24.15 secretion, yet little is understood concerning constitutive release of the peptidase. Utilizing the mouse hypothalamic neuronal GT1-7 cell line, we demonstrate that EP24.15 exists within lipid rafts in the plasma membrane, and that the enzyme is localized to the exofacial leaflet of lipid rafts. Further, we have found that biotinylated EP24.15 on the extracellular surface is released into the cell media in a fashion similar to constitutive release. In addition, classical and non-classical secretion pathway inhibitors were employed to understand the release of EP24.15 into surrounding cell media. The non-classical secretion inhibitor glyburide, a blocker of ATP-sensitive K+ channels, decreased the amount of constitutively released EP24.15 in cell media of GT1-7 cells. With these data, we conclude that EP24.15 association with lipid rafts on the extracellular surface precedes constitutive release of the peptidase into the extracellular milieu for its action on neuropeptides.

Biotinylation↗

PubMed2004

The role of neuropeptide processing enzymes in endocrine (prostate) cancer: EC 3.4.24.15 (EP24.15).

The zinc metalloendopeptidase EC3.4.24.15 [EP24.15, thimet oligopeptidase], a neuropeptide processing enzyme, is central to the formation and degradation of many bioactive peptides in the neural proteome, and is highly expressed in normal prostate. EP24.15 actions are increased in androgen-dependent prostate cancer compared to androgen-independent; augmented by androgen treatment, and inhibited by clinical GnRH analogs. The "neural" prostate includes: neuropeptides, cognate receptors and processing enzymes regulating signaling of peptide-mediated neural inputs.

Humans↗

PubMed2003

EP24.15 is associated with lipid rafts.

Metalloendopeptidase EC 3.4.24.15 (EP24.15, thimet oligopeptidase) is a neuropeptide-metabolizing peptidase expressed throughout the body, but primarily in the brain, gonads, and pituitary. For EP24.15 to have its greatest effect upon peptides in the periphery, it must be targeted and released into the extracellular space. Western blot analysis of fractions taken from discontinuous sucrose density gradients carried out on crude plasma membrane fractions from AtT-20 cells reveals colocalization of EP24.15 and flotillin-1, a known lipid raft marker. Further analysis revealed that an intracellular membrane marker and non-lipid raft, plasma membrane marker, failed to colocalize, supporting EP24.15/lipid raft association. Furthermore, EP24.15 immunoreactivity in lipid raft fractions generated from cells treated with methyl beta-cyclodextrin (MbetaCD) was greatly reduced. Finally, treatment with MbetaCD resulted in the accumulation of EP24.15 in the media of drug-treated cells over vehicle-treated cells, suggesting that a large percentage of EP24.15 associating with lipid rafts resides on the extracellular surface of the plasma membrane. With this exofacial localization, EP24.15 could have ample access to neuropeptides not only in the immediate microenvironment, but the ability to degrade or modify peptides bound for receptor interaction.

Animals↗

PubMed2003

Regulation of cell-surface major histocompatibility complex class I expression by the endopeptidase EC3.4.24.15 (thimet oligopeptidase).

Endopeptidase EP24.15 (EC 3.4.24.15; thimet oligopeptidase), traditionally classified as a neuropeptide-processing enzyme, degrades well-known MHC I (major histocompatibility complex class I) peptides in cell extracts. In the present study, we determine the contribution of EP24.15 in vivo to the surface expression of MHC I on intact cells. CTLs (cytotoxic T-lymphocytes) recognize a vast array of peptides presented in the context of MHC I cell-surface molecules. Stable retroviral overexpression of EP24.15 induces a dramatic, long-term inhibition of surface MHC I. In contrast, overexpression of a mutant EP24.15, which is expressed, but is enzymically inactive, does not affect the surface MHC I expression level. We observed no difference in the effect of EP24.15 on the expression of different classes of MHC I. IFN-gamma (interferon-gamma) treatment re-established MHC I expression on these EP24.15-overexpressing cells, and also induced EP24.15 cytosolic protein expression and enzyme activity. To our knowledge, this is the first demonstration of cytokine-induced EP24.15 expression and activity. Conversely, stable retroviral silencing of endogenous EP24.15 by RNA interference induced a striking, long-term increase in surface MHC I. Subcellular fractionation and enzyme-activity experiments localized the vast majority of EP24.15 protein expression and function to the cytosol. Therefore we introduce a novel function of the cytosolic form of EP24.15. EP24.15 activity in the extracellular space is significant for neuropeptide processing, and in the present paper, we demonstrate that EP24.15 activity in the cytosol may be significant for regulation of MHC I cell-surface expression.

Animals↗

PubMed2003

Mutations in capillary morphogenesis gene-2 result in the allelic disorders juvenile hyaline fibromatosis and infantile systemic hyalinosis.

Juvenile hyaline fibromatosis (JHF) and infantile systemic hyalinosis (ISH) are autosomal recessive syndromes of unknown etiology characterized by multiple, recurring subcutaneous tumors, gingival hypertrophy, joint contractures, osteolysis, and osteoporosis. Both are believed to be allelic disorders; ISH is distinguished from JHF by its more severe phenotype, which includes hyaline deposits in multiple organs, recurrent infections, and death within the first 2 years of life. Using the previously reported chromosome 4q21 JHF disease locus as a guide for candidate-gene identification, we identified and characterized JHF and ISH disease-causing mutations in the capillary morphogenesis factor-2 gene (CMG2). Although CMG2 encodes a protein upregulated in endothelial cells during capillary formation and was recently shown to function as an anthrax-toxin receptor, its physiologic role is unclear. Two ISH family-specific truncating mutations, E220X and the 1-bp insertion P357insC that results in translation of an out-of-frame stop codon, were generated by site-directed mutagenesis and were shown to delete the CMG-2 transmembrane and/or cytosolic domains, respectively. An ISH compound mutation, I189T, is predicted to create a novel and destabilizing internal cavity within the protein. The JHF family-specific homoallelic missense mutation G105D destabilizes a von Willebrand factor A extracellular domain alpha-helix, whereas the other mutation, L329R, occurs within the transmembrane domain of the protein. Finally, and possibly providing insight into the pathophysiology of these diseases, analysis of fibroblasts derived from patients with JHF or ISH suggests that CMG2 mutations abrogate normal cell interactions with the extracellular matrix.

Amino Acid Sequence↗

PubMed2003

pH dependence studies provide insight into the structure and mechanism of thimet oligopeptidase (EC 3.4.24.15).

Thimet oligopeptidase (EC 3.4.24.15; TOP) is a Zn(II) endopeptidase implicated in physiological regulation of processes involving neuropeptides. The present study clarifies the active site structure and mechanism of catalysis of TOP. The enzyme exhibited a bell-shaped pH dependence of activity having an acidic limb due to a protonation event with a pK(a) of 5.7 and a basic limb with pK(a) of 8.8. The acidic limb can be attributed to protonation of a residue affecting k(cat) while the alkaline limb may be due to conformational change. Mutation of Tyr612 to Phe resulted in more than 400-fold decrease in activity. This result, supported by modeling studies, implicates Tyr612 in transition state stabilization analogous to the role of His231 of thermolysin.

Animals↗

PubMed2003

Proteomics in the discovery of new therapeutic targets for psychiatric disease.

In terms of impact on and cost to society psychiatric disorders are among the most important health problems of today. Current estimates from the US suggest that the collective cost of psychiatric diseases could amount to one-third of the total health care budget with a cumulative lifetime prevalence of 30%. While undoubtedly improvements have been made in the diagnosis and treatment of at least the symptoms of mental illness, there has been frustratingly little progress in elucidating the molecular mechanisms. However, a fundamentally different approach to study molecular mechanisms of psychiatric diseases is emerging as a result of technological advances in expression profiling methods. This comprises the investigation of the expressed disease 'phenotypes', developing from the differential gene and protein expression in the central nervous system as a result of the complex interaction between genetic predisposition and environmental modulation. This paper will focus on proteomics, expression profiling at the protein level, reviewing some of the available tools and their application in the molecular analysis of psychiatric disease.

Antipsychotic Agents↗