Biomedical subjects

Emer S Ferro

Publications and source records attributed to Emer S Ferro.

14 recordsLinked to original sources

PubMed2006

Conformation state-sensitive antibodies to G-protein-coupled receptors.

A growing body of evidence indicates that G-protein-coupled receptors undergo complex conformational changes upon agonist activation. It is likely that the extracellular region, including the N terminus, undergoes activation-dependent conformational changes. We examined this by generating antibodies to regions within the N terminus of micro-opioid receptors. We find that antibodies to the midportion of the N-terminal tail exhibit enhanced recognition of activated receptors, whereas those to the distal regions do not. The enhanced recognition is abolished upon treatment with agents that block G-protein coupling or deglycosylate the receptor. This suggests that the N-terminal region of mu receptors undergoes conformational changes following receptor activation that can be selectively detected by these region-specific antibodies. We used these antibodies to characterize micro receptor type-specific ligands and find that the antibodies accurately differentiate ligands with varying efficacies. Next, we examined if these antibodies can be used to investigate the extent and duration of activation of endogenous receptors. We find that peripheral morphine administration leads to a time-dependent increase in antibody binding in the striatum and prefrontal cortex with a peak at about 30 min, indicating that these antibodies can be used to probe the spatio-temporal dynamics of native mu receptors. Finally, we show that this strategy of targeting the N-terminal region to generate receptor conformation-specific antisera can be applied to other G(alpha)(i)-coupled (delta-opioid, CB1 cannabinoid, alpha(2A)-adrenergic) as well as G(alpha)(s)-(beta(2)-adrenergic) and G(alpha)(q)-coupled (AT1 angiotensin) receptors. Taken together, these studies describe antisera as tools that allow, for the first time, studies probing differential conformation states of G-protein-coupled receptors, which could be used to identify molecules of therapeutic interest.

Analgesics, Opioid↗

PubMed2006

Role of the Cys18-Cys274 disulfide bond and of the third extracellular loop in the constitutive activation and internalization of angiotensin II type 1 receptor.

An insertion of residues in the third extracellular loop and a disulfide bond linking this loop to the N-terminal domain were identified in a structural model of a G-protein coupled receptor specific to angiotensin II (AT1 receptor), built in homology to the seven-transmembrane-helix bundle of rhodopsin. Both the insertion and the disulfide bond were located close to an extracellular locus, flanked by the second extracellular loop (EC-2), the third extracellular loop (EC-3) and the N-terminal domain of the receptor; they contained residues identified by mutagenesis studies to bind the angiotensin II N-terminal segment (residues D1 and R2). It was postulated that the insertion and the disulfide bond, also found in other receptors such as those for bradykinin, endothelin, purine and other ligands, might play a role in regulating the function of the AT1 receptor. This possibility was investigated by assaying AT1 forms devoid of the insertion and with mutations to Ser on both positions of Cys residues forming the disulfide bond. Binding and activation experiments showed that abolition of this bond led to constitutive activation, decay of agonist binding and receptor activation levels. Furthermore, the receptors thus mutated were translocated to cytosolic environments including those in the nucleus. The receptor form with full deletion of the EC-3 loop residue insertion, displayed a wild type receptor behavior.

Amino Acid Sequence↗

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

A role for transmembrane domains V and VI in ligand binding and maturation of the angiotensin II AT1 receptor.

Several studies have proposed that angiotensin II (Ang II) binds to its receptor AT1 through interactions with residues in helices V and VI, suggesting that the distance between these helices is crucial for ligand binding. Based on a 3D model of AT1 in which the C-terminus of Ang II is docked, we identified the hydrophobic residues of TM V and VI pointing towards the external face of the helices, which may play a role in the structure of the binding pocket and in the structural integrity of the receptor. We performed a systematic mutagenesis study of these residues and examined the binding, localization, maturation, and dimerization of the mutated receptors. We found that mutations of hydrophobic residues to alanine in helix V do not alter binding, whereas mutations to glutamate lead to loss of binding without a loss in cell surface expression, suggesting that the external face of helix V may not directly participate in binding, but may rather contribute to the structure of the binding pocket. In contrast, mutations of hydrophobic residues to glutamate in helix VI lead to a loss in cell surface expression, suggesting that the external surface of helix VI plays a structural role and ensures correct folding of the receptor.

Alanine↗

PubMed2005

14-3-3 epsilon modulates the stimulated secretion of endopeptidase 24.15.

Endopeptidase 24.15 (ep24.15: EC3.4.24.15), a secreted protein involved in peptide metabolism, is unusual in that it does not contain a signal peptide sequence. In this work, we describe the physical interaction between ep24.15 and 14-3-3 epsilon, one isoform of a family of ubiquitous phosphoserine/threonine-scaffold proteins that organizes cell signaling and is involved in exocytosis. The interaction between ep24.15 and 14-3-3 epsilon increased following phosphorylation of ep24.15 at Ser(644) by protein kinase A (PKA). The co-localization of ep24.15 and 14-3-3 epsilon was increased by exposure of HEK293 cells (human embryonic kidney cells) to forskolin (10 microm). Overexpression of 14-3-3 epsilon in HEK293 cells almost doubled the secretion of ep24.15 stimulated by A23187 (7.5 microm) from 10%[1.4 +/- 0.24 AFU/(min 10(6) cells)] to 19%[2.54 +/- 0.24 AFU/(min 10(6) cells)] (p < 0.001) of the total intracellular enzyme activity. Treatment with forskolin had a synergistic effect on the A23187-stimulated secretion of ep24.15 that was totally blocked by the PKA inhibitor KT5720. The ep24.15 point mutation S644A reduced the co-localization of ep24.15 and 14-3-3 in stably transfected HEK293 cells. Indeed, secretion of the ep24.15 S644A mutant from these cells was only slightly stimulated by A23187 and insensitive to forskolin, in contrast to that of the wild type enzyme. Together, these data suggest that prior interaction with 14-3-3 is an important step in the unconventional stimulated secretion of ep24.15.

14-3-3 Proteins↗

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

A transcript finishing initiative for closing gaps in the human transcriptome.

We report the results of a transcript finishing initiative, undertaken for the purpose of identifying and characterizing novel human transcripts, in which RT-PCR was used to bridge gaps between paired EST clusters, mapped against the genomic sequence. Each pair of EST clusters selected for experimental validation was designated a transcript finishing unit (TFU). A total of 489 TFUs were selected for validation, and an overall efficiency of 43.1% was achieved. We generated a total of 59,975 bp of transcribed sequences organized into 432 exons, contributing to the definition of the structure of 211 human transcripts. The structure of several transcripts reported here was confirmed during the course of this project, through the generation of their corresponding full-length cDNA sequences. Nevertheless, for 21% of the validated TFUs, a full-length cDNA sequence is not yet available in public databases, and the structure of 69.2% of these TFUs was not correctly predicted by computer programs. The TF strategy provides a significant contribution to the definition of the complete catalog of human genes and transcripts, because it appears to be particularly useful for identification of low abundance transcripts expressed in a restricted set of tissues as well as for the delineation of gene boundaries and alternatively spliced isoforms.

Alternative Splicing↗

PubMed2004

The genome sequence of the gram-positive sugarcane pathogen Leifsonia xyli subsp. xyli.

The genome sequence of Leifsonia xyli subsp. xyli, which causes ratoon stunting disease and affects sugarcane worldwide, was determined. The single circular chromosome of Leifsonia xyli subsp. xyli CTCB07 was 2.6 Mb in length with a GC content of 68% and 2,044 predicted open reading frames. The analysis also revealed 307 predicted pseudogenes, which is more than any bacterial plant pathogen sequenced to date. Many of these pseudogenes, if functional, would likely be involved in the degradation of plant heteropolysaccharides, uptake of free sugars, and synthesis of amino acids. Although L. xyli subsp. xyli has only been identified colonizing the xylem vessels of sugarcane, the numbers of predicted regulatory genes and sugar transporters are similar to those in free-living organisms. Some of the predicted pathogenicity genes appear to have been acquired by lateral transfer and include genes for cellulase, pectinase, wilt-inducing protein, lysozyme, and desaturase. The presence of the latter may contribute to stunting, since it is likely involved in the synthesis of abscisic acid, a hormone that arrests growth. Our findings are consistent with the nutritionally fastidious behavior exhibited by L. xyli subsp. xyli and suggest an ongoing adaptation to the restricted ecological niche it inhabits.

Actinomycetales↗

PubMed2004

Intracellullar peptides as putative natural regulators of protein interactions.

Extralysosomal proteolysis by multicatalytic complexes such as the 26S proteasome produces large amounts of peptides in the cytosol, mitochondria and nuclei of eukaryotic cells, and there is increasing evidence that the resulting free intracellular peptides can modulate specific protein interactions. The demonstration that free peptides added to the intracellular milieu can regulate cellular functions mediated by protein interactions suggests new putative roles for these molecules in gene regulation, metabolism, cell signaling and protein targeting. Such interactions frequently involve specific consensus amino acid sequences that can be predicted based on similarities in domain composition. We have recently developed a new strategy for identifying novel natural peptides, the sequences of which correspond to fragments of intracellular proteins and contain putative post-translational modification sites. In this review, we examine the evidence that intracellular peptides released by proteasomes may be involved in regulating protein interactions. In particular, the role of endopeptidase 24.15 (thimet oligopeptidase; EC 3.4.24.15) is discussed in detail as this enzyme has been implicated in intracellular peptide metabolism in vivo in concert with the 26S proteasome.

Animals↗

PubMed2004

The intracellular distribution and secretion of endopeptidases 24.15 (EC 3.4.24.15) and 24.16 (EC 3.4.24.16).

Endopeptidase 24.15 (EC 3.4.24.15; EP24.15) and endopeptidase 24.16 (EC 3.4.24.16; EP24.16) are enzymes involved in general peptide metabolism in mammalian cells and tissues. This review will focus on morphological and biochemical aspects related to the subcellular distribution and secretion of these homologous enzymes in the central nervous system. These are important issues for a better understanding of the functions of EP24.15 and EP24.16 within neuroendocrine systems.

Animals↗

PubMed2003

A structure-based site-directed mutagenesis study on the neurolysin (EC 3.4.24.16) and thimet oligopeptidase (EC 3.4.24.15) catalysis.

Neurolysin (EP24.16) and thimet oligopeptidase (EP24.15) are closely related metalloendopeptidases. Site-directed mutagenesis of Tyr(613) (EP24.16) or Tyr(612) (EP24.15) to either Phe or Ala promoted a strong reduction of k(cat)/K(M) for both enzymes. These data suggest the importance of both hydroxyl group and aromatic ring at this specific position during substrate hydrolysis by these peptidases. Furthermore, the EP24.15 A607G mutant showed a k(cat)/K(M) of 2x10(5) M(-1) s(-1) for the Abz-GFSIFRQ-EDDnp substrate, similar to that of EP24.16 (k(cat)/K(M)=3x10(5) M(-1) s(-1)) which contains Gly at the corresponding position; the wild type EP24.15 has a k(cat)/K(M) of 2.5x10(4) M(-1) s(-1) for this substrate.

Alanine↗

PubMed2002

Novel natural peptide substrates for endopeptidase 24.15, neurolysin, and angiotensin-converting enzyme.

Endopeptidase 24.15 (EC; ep24.15), neurolysin (EC; ep24.16), and angiotensin-converting enzyme (EC; ACE) are metallopeptidases involved in neuropeptide metabolism in vertebrates. Using catalytically inactive forms of ep24.15 and ep24.16, we have identified new peptide substrates for these enzymes. The enzymatic activity of ep24.15 and ep24.16 was inactivated by site-directed mutagenesis of amino acid residues within their conserved HEXXH motifs, without disturbing their secondary structure or peptide binding ability, as shown by circular dichroism and binding assays. Fifteen of the peptides isolated were sequenced by electrospray ionization tandem mass spectrometry and shared homology with fragments of intracellular proteins such as hemoglobin. Three of these peptides (PVNFKFLSH, VVYPWTQRY, and LVVYPWTQRY) were synthesized and shown to interact with ep24.15, ep24.16, and ACE, with K(i) values ranging from 1.86 to 27.76 microm. The hemoglobin alpha-chain fragment PVNFKFLSH, which we have named hemopressin, produced dose-dependent hypotension in anesthetized rats, starting at 0.001 microg/kg. The hypotensive effect of the peptide was potentiated by enalapril only at the lowest peptide dose. These results suggest a role for hemopressin as a vasoactive substance in vivo. The identification of these putative intracellular substrates for ep24.15 and ep24.16 is an important step toward the elucidation of the role of these enzymes within cells.

Amino Acid Sequence↗

PubMed2002

Differential expression of glycosaminoglycans and proteoglycans in the migratory pathway of the primordial germ cells of the mouse.

Primordial germ cells are an embryonic cell line that give rise to gametes in vertebrates. They originate outside the embryo proper and migrate by a well-defined route to the genital ridges. Proteoglycans and glycosaminoglycans have distinctive properties that affect many of the characteristics of the extracellular microenvironment of migratory pathways in a variety of developmental systems. The purpose of this work was to identify the proteoglycans and glycosaminoglycans that are spatially and temporally expressed in the migratory pathway of primordial germ cells. We showed that the expression of proteoglycans and glycosaminoglycans in the primordial germ cells migratory pathway changes according to the different phases of the migratory process. Some molecules such as chondroitin-0-sulfate, decorin, and biglycan are present only in certain phases of the migratory process of primordial germ cells. Heparan sulfate, chondroitin-6-sulfate, versican, perlecan, and syndecan-4, although exhibiting some variation in expression were detected during all phases of the migratory process. Our results indicate that the successive steps of primordial germ cell migration require a coordinated expression of proteoglycans and glycosaminoglycans, that should be present in appropriate levels and in specific areas of the embryo, and that the sequential expression of these extracellular matrix molecules is under a genetic program that appears to be common to a variety of cell types during embryonic development.

Animals↗

PubMed2002

Temperature and salts effects on the peptidase activities of the recombinant metallooligopeptidases neurolysin and thimet oligopeptidase.

We report the recombinant neurolysin and thimet oligopeptidase (TOP) hydrolytic activities towards internally quenched fluorescent peptides derived from the peptide Abz-GGFLRRXQ-EDDnp (Abz, ortho-aminobenzoicacid; EDDnp, N-(2,4-dinitrophenyl) ethylenediamine), in which X was substituted by 11 different natural amino acids. Neurolysin hydrolyzed these peptides at R-R or at R-X bonds, and TOP hydrolyzed at R-R or L-R bonds, showing a preference to cleave at three or four amino acids from the C-terminal end. The kinetic parameters of hydrolysis and the variations of the cleavage sites were evaluated under different conditions of temperature and salt concentration. The relative amount of cleavage varied with the nature of the substitution at the X position as well as with temperature and NaCl concentration. TOP was activated by all assayed salts in the range 0.05-0.2 m for NaCl, KCl, NH4Cl and NaI, and 0.025-0.1 m for Na2SO4. Concentration higher than 0.2 N NH4Cl and NaI reduced TOP activity, while 0.5 N or higher concentration of NaCl, KCl and Na2SO4 increased TOP activity. Neurolysin was strongly activated by NaCl, KCl and Na2SO4, while NH4Cl and NaI have very modest effect. High positive values of enthalpy (DeltaH*) and entropy (DeltaS*) of activation were found together with an unusual temperature dependence upon the hydrolysis of the substrates. The effects of low temperature and high NaCl concentration on the hydrolytic activities of neurolysin and TOP do not seem to be a consequence of large secondary structure variation of the proteins, as indicated by the far-UV CD spectra. However, the modulation of the activities of the two oligopeptidases could be related to variations of conformation, in limited regions of the peptidases, enough to modify their activities.

Amino Acids↗