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1.
Diabetologia ; 55(11): 2989-98, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22898766

ABSTRACT

AIMS/HYPOTHESIS: A progressive loss of pancreatic beta cell function, a decrease in beta cell mass and accumulation of islet amyloid is characteristic of type 2 diabetes mellitus. The main constituent of islet amyloid is islet amyloid polypeptide (IAPP). In this study, we examined the ability of the peptidase neprilysin to cleave IAPP and prevent human IAPP-induced pancreatic beta cell toxicity. METHODS: Neprilysin and a catalytically compromised neprilysin mutant were tested for their ability to inhibit human IAPP fibrillisation and human IAPP-induced pancreatic beta cell cytotoxicity. Degradation of human IAPP by neprilysin was followed by HPLC, and the degradation products were identified by MS. RESULTS: Neprilysin prevented IAPP fibrillisation by cleaving IAPP at Arg(11)-Leu(12), Leu(12)-Ala(13), Asn(14)-Phe(15), Phe(15)-Leu(16), Asn(22)-Phe(23) and Ala(25)-Ile(26). It also appears to prevent human IAPP fibrillisation through a non-catalytic interaction. Neprilysin protected against beta cell cytotoxicity induced by exogenously added or endogenously produced human IAPP. CONCLUSIONS/INTERPRETATION: The data presented support a potential therapeutic role for neprilysin in preventing type 2 diabetes mellitus. This study supports the hypothesis that extracellular human IAPP contributes to human IAPP-induced beta cell cytotoxicity. Whether human IAPP exerts its cytotoxic effect through a totally extracellular mechanism or through a cellular reuptake mechanism is unclear at this time.


Subject(s)
Amyloid beta-Protein Precursor/metabolism , Apoptosis/physiology , Diabetes Mellitus, Type 2/metabolism , Insulin-Secreting Cells/enzymology , Neprilysin/metabolism , Amino Acid Sequence , Amyloid/metabolism , Amyloid beta-Protein Precursor/genetics , Animals , Apoptosis/drug effects , Cell Line, Tumor , Diabetes Mellitus, Type 2/pathology , Enzyme Activation/physiology , Green Fluorescent Proteins/genetics , Humans , Insulin-Secreting Cells/drug effects , Insulinoma , Molecular Sequence Data , Neprilysin/genetics , Neprilysin/pharmacology , Pancreatic Neoplasms , Rats , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Substrate Specificity/physiology , Transfection
2.
Prostate Cancer Prostatic Dis ; 11(1): 79-87, 2008.
Article in English | MEDLINE | ID: mdl-17563767

ABSTRACT

Neutral endopeptidase (NEP) is a cell surface peptidase that catalytically inactivates a variety of physiologically active peptides including basic fibroblast growth factor (FGF-2). We investigated the effect of using lentivirus to overexpress NEP in NEP-deficient DU145 prostate cancer cells. Third-generation lentiviral vectors encoding wild-type NEP (L-NEP), catalytically inactive mutant NEP (L-NEPmu), and green fluorescent protein (L-GFP) were stably introduced into DU145 cells. FGF-2 levels in cell culture supernatants decreased by 80% in L-NEP-infected DU145 cells compared to cells infected with L-NEPmu or L-GFP (P<0.05) while levels of other angiogenic factors were not altered. In vitro tubulogenesis of human vascular endothelial cells induced by conditioned media from DU145 cells infected with L-NEP was significantly reduced compared with that from DU145 cells infected with L-GFP (P<0.05). Tumor xenografts from L-NEP-infected DU145 cells were significantly smaller compared to control cell xenografts and vascularity within these tumors was decreased (P<0.05). Our data suggest that stable expression of NEP in DU145 cells inhibits prostate cancer tumorigenicity by inhibiting angiogenesis, with a probable mechanism being proteolytic inactivation of FGF-2.


Subject(s)
Fibroblast Growth Factor 2/metabolism , Neovascularization, Pathologic/prevention & control , Neprilysin/metabolism , Prostatic Neoplasms/blood supply , Prostatic Neoplasms/therapy , Animals , Cell Proliferation , Cells, Cultured , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Genetic Vectors , Humans , Immunoblotting , Immunoprecipitation , Lentivirus/genetics , Male , Mice , Mice, Nude , Neoplasm Invasiveness , Neprilysin/genetics , Prostatic Neoplasms/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Xenograft Model Antitumor Assays
3.
Cancer Gene Ther ; 14(6): 583-9, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17415380

ABSTRACT

Neprilysin (neutral endopeptidase, NEP) is a cell surface peptidase whose expression is lost in approximately 50% of prostate cancers (PC). NEP normally functions to inactivate peptides such as bombesin and endothelin-1, and potentiates the effects of the PTEN tumor suppressor via a direct protein-protein interaction. NEP loss contributes to PC progression. We investigated the therapeutic efficacy of using a lentiviral vector system to restore NEP expression in PC cells. Third-generation lentiviral vectors encoding wild-type NEP (L-NEP) or green fluorescent protein (L-GFP) were introduced into NEP-deficient 22RV1 PC cells. Cells infected with L-NEP or L-GFP at a multiplicity of infection of 10 demonstrated NEP enzyme activity of 1171.2+/-4.9 and 17.2+/-5.3 pmol/microg/min (P<0.0001), respectively. Cell viability, proliferation and invasion were each significantly inhibited in 22RV1 cells expressing NEP compared with control cells infected with L-GFP (P<0.01). Analysis of known downstream effects of NEP showed NEP-expressing cells exhibiting decreased Akt and focal adhesion kinase phosphorylation and increased PTEN protein expression. Finally, injection of L-NEP into established 22RV1 xenograft tumors significantly inhibited tumor growth (P<0.01). These experiments demonstrate that lentiviral NEP gene transfer is a novel targeted strategy for the treatment of NEP-deficient PC.


Subject(s)
Lentivirus/genetics , Neoplasm Invasiveness/physiopathology , Neprilysin/therapeutic use , Prostatic Neoplasms/therapy , Animals , Cell Line , Disease Models, Animal , Gene Transfer Techniques , Genetic Vectors , Humans , Male , Mice , Mice, Nude , Neprilysin/genetics , Neprilysin/immunology , Prostatic Neoplasms/immunology , Xenograft Model Antitumor Assays
5.
Neuroscience ; 120(1): 249-59, 2003.
Article in English | MEDLINE | ID: mdl-12849757

ABSTRACT

A significant number of the cholinergic neurons in the basal forebrain of the primate, but not the rodent brain contain the calcium binding protein calbindin-D28k (CB). Previous experiments in our laboratory have demonstrated a substantial age-related loss of CB from the human basal forebrain cholinergic neurons (BFCN). The present study investigated the possible age-related loss of CB from the BFCN in a non-human primate species, the common marmoset (Callithrix jacchus). Quantitative analysis of matching sections as well as unbiased stereological determination of neuronal number were used in 16 adult marmosets ranging in age between 2 and 15 years. No significant changes were observed in the number of choline acetyltransferase-positive BFCN when a group of young animals (< or =4 years) was compared with a 6-8-year-old group and a 9-15-year-old group. Similarly, no age-related changes were observed in Nissl-stained magnocellular basal forebrain (putatively cholinergic) neurons. In contrast, the BFCN of the two older groups of animals displayed a significant loss of CB. The age-related loss of CB occurred in all sectors of the BFCN, but was greatest in the anterior sector of this cell group. The CB loss was neurochemically specific since the BFCN in the older groups of animals continued to express other markers such as high and low affinity neurotrophin receptors. The age-related loss of CB from the marmoset BFCN was also regionally selective as CB positive neurons in other structures, such as the cerebral cortex and the striatum displayed no apparent age-related changes. These results indicate that the marmoset BFCN display a significant and selective age-related loss of CB reminiscent of that observed in the human. Therefore, the common marmoset represents an appropriate animal model in which the consequences of BFCN CB loss can be investigated in depth. Loss of CB from the aged BFCN is likely to reduce the capacity of these neurons to buffer intracellular calcium and to leave them vulnerable to insults which can result in increased calcium levels. The vulnerability of the CB-negative BFCN in the aged marmoset to various insults which disturb calcium homeostasis remains to be investigated.


Subject(s)
Aging/metabolism , Cholinergic Fibers/metabolism , Prosencephalon/metabolism , S100 Calcium Binding Protein G/metabolism , Aging/pathology , Animals , Calbindin 1 , Calbindins , Callithrix , Cholinergic Fibers/chemistry , Cholinergic Fibers/pathology , Prosencephalon/chemistry , Prosencephalon/pathology , S100 Calcium Binding Protein G/analysis
6.
Biochemistry ; 40(31): 9447-52, 2001 Aug 07.
Article in English | MEDLINE | ID: mdl-11478915

ABSTRACT

Kinetic evidence suggests an acidic region in nardilysin binds polyamines and acts as a regulatory domain. The binding of approximately 5 mol of spermine/mol of nardilysin was demonstrated. The binding curve was sigmoidal exhibiting an IC(50) of approximately 118 microM and a Hill coefficient of 1.8. Spermine diminished the tryptophan fluorescence of the enzyme and increased its sensitivity to protease V8. The acidic stretch from mouse and human nardilysin were expressed as glutathione transferase fusion proteins. All fusion proteins bound spermine with an IC(50) of 40 to 110 microM. The mouse fusion protein bound approximately 7 mol of spermine exhibiting a sigmoidal binding curve and a Hill coefficient of 1.4. The human acidic stretch, containing fewer acidic residues, bound approximately 5 mol of spermine/mol with a hyperbolic binding curve. Chimeric fusion proteins containing the N-terminus of the mouse acidic region fused to the C-terminus of the human acidic region bound approximately 10 mol of spermine, while the opposite chimera bound approximately 4 mol of spermine/mol. The N-terminal region of the mouse acidic domain binds 3--4 mol spermine/mol exhibiting a Hill coefficient of 1.4, while the same region from human nardilysin binds 1 mol of spermine/mol. Spermine enhanced the sensitivity of the mouse acidic domain, but not the human acidic domain, to protease V8. Together the data support a model where the acidic stretch of nardilysin functions as an autonomous domain.


Subject(s)
Metalloendopeptidases/metabolism , Peptide Fragments/metabolism , Amino Acid Sequence , Animals , Glutathione Transferase/genetics , Humans , Hydrogen-Ion Concentration , Kinetics , Metalloendopeptidases/chemistry , Metalloendopeptidases/genetics , Mice , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/genetics , Protein Binding/genetics , Protein Conformation , Protein Structure, Tertiary/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/metabolism , Spermine/metabolism
7.
J Biol Chem ; 276(16): 13121-6, 2001 Apr 20.
Article in English | MEDLINE | ID: mdl-11145971

ABSTRACT

The transcription factor neuron-restrictive silencer factor/repressor element-1 (RE-1) silencing transcription factor (NRSF/REST) contains nine zinc finger domains and binds to the DNA element, neuron-restrictive silencer element/repressor element-1. REST4, a C-terminally truncated form of NRSF/REST, contains the five N-terminal zinc fingers and binds weakly to DNA yet is transported into the nucleus. To study the contribution of zinc fingers 6-8 to DNA binding, each was mutated. A mutation in zinc finger 6 or 8 had little effect; however, mutation of zinc finger 7 diminished DNA binding. Mutations in any two of these zinc fingers eliminated DNA binding. The contribution of zinc fingers 2-5 to nuclear targeting was studied. Deletion of zinc finger 5 prevented nuclear targeting. Mutations in zinc finger 2, 4, or 5 did not abolish nuclear targeting. However, a zinc finger 3 mutation together with a zinc finger 2 mutation localized to the nuclear envelope. A zinc finger 3 mutation alone or in combination with a zinc finger 4 or 5 mutation produced a punctate nuclear distribution. These results suggest the presence of signals for nuclear targeting, for nuclear entry, and for release from the translocation machinery within zinc fingers 2-5 of REST4.


Subject(s)
Cell Nucleus/metabolism , DNA/metabolism , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism , Base Sequence , Binding Sites , Cell Line , DNA/chemistry , DNA Primers , Green Fluorescent Proteins , Humans , Luminescent Proteins/analysis , Molecular Sequence Data , Mutagenesis , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Deletion , Templates, Genetic , Transfection , Zinc Fingers , beta-Galactosidase/analysis
8.
J Biol Chem ; 276(2): 1152-5, 2001 Jan 12.
Article in English | MEDLINE | ID: mdl-11042190

ABSTRACT

Recombinant rat insulysin was shown to cleave the internally quenched fluorogenic peptide 2-aminobenzyl-GGFLRKVGQ-ethylenediamine-2,4-dinitrophenol at the R-K bond, exhibiting a K(m) of 13 microm and a V(max) of 2.6 micromol min(-1) mg(-1). Derivatives of this peptide in which the P(2) leucine or the P(2)' valine were replaced with other residues were used to probe the subsite specificity of the enzyme. Varying the P(2) residue produced a 4-fold range in K(m) and a 7-fold range in k(cat). The nature of the P(2) residue had a significant effect on the site of cleavage. Leucine, isoleucine, valine, and aspartate produced cleavage at the R-K bond. Asparagine produced 36% cleavage at the N-R bond and 64% cleavage at the R-K bond, whereas with alanine or serine the A-R and S-R bonds were the major cleavage sites. With tyrosine, phenylalanine, methionine, or histidine representing the varied residue X, cleavages at F-X, X-R, and R-K were seen, whereas with tryptophan equal cleavage occurred at the F-W and W-R bonds. Variable P(2)' residues produce less of a change in both K(m) and k(cat) and have little influence on the cleavage site. Exceptions are phenylalanine, tyrosine, leucine, and isoleucine, which in addition to producing cleavage at the R-K bond, produce significant cleavage at the L-R bond. Alanine and tyrosine were unique in producing cleavage at the F-L bond. Taken together, these data suggest that insulysin specificity is directed toward the amino side of hydrophobic and basic residues and that the enzyme has an extended substrate binding site.


Subject(s)
Dinitrophenols/pharmacokinetics , Insulysin/metabolism , Oligopeptides/pharmacokinetics , Amino Acid Sequence , Animals , Binding Sites , Chromatography, High Pressure Liquid , Hydrolysis , Kinetics , Oligopeptides/chemistry , Oligopeptides/metabolism , Rats , Recombinant Proteins/metabolism , Substrate Specificity
9.
J Neurosci ; 20(23): 8745-9, 2000 Dec 01.
Article in English | MEDLINE | ID: mdl-11102481

ABSTRACT

Insulysin (EC. 3.4.22.11) has been implicated in the clearance of beta amyloid peptides through hydrolytic cleavage. To further study the action of insulysin on Abeta peptides recombinant rat insulysin was used. Cleavage of both Abeta(1-40) and Abeta(1-42) by the recombinant enzyme was shown to initially occur at the His(13)-His(14), His(14)-Gln(15), and Phe(19)-Phe(20) bonds. This was followed by a slower cleavage at the Lys(28)-Gly(29), Val(18)-Phe(19), and Phe(20)-Ala(21) positions. None of the products appeared to be further metabolized by insulysin. Using a rat cortical cell system, the action of insulysin on Abeta(1-40) and Abeta(1-42) was shown to eliminate the neurotoxic effects of these peptides. Insulysin was further shown to prevent the deposition of Abeta(1-40) onto a synthetic amyloid. Taken together these results suggest that the use of insulysin to hydrolyze Abeta peptides represents an alternative gene therapeutic approach to the treatment of Alzheimer's disease.


Subject(s)
Amyloid beta-Peptides/metabolism , Insulysin/metabolism , Peptide Fragments/metabolism , Plaque, Amyloid/metabolism , Amyloid beta-Peptides/chemistry , Animals , Cell Survival/drug effects , Cells, Cultured , Chromatography, High Pressure Liquid , Hydrolysis , Insulysin/chemistry , Insulysin/genetics , Insulysin/pharmacology , Neurons/cytology , Neurons/drug effects , Neurons/metabolism , Neuroprotective Agents/metabolism , Neuroprotective Agents/pharmacology , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Plaque, Amyloid/chemistry , Rats , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
10.
Acta Crystallogr D Biol Crystallogr ; 56(Pt 12): 1644-6, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11092934

ABSTRACT

Neuropeptidases inactivate or modify the activity of peptide neurotransmitters and neurohormones. The neuropeptidase neurolysin acts only on short peptides and accepts a variety of cleavage-site sequences. Structures of the enzyme and enzyme-substrate complexes will help to determine the mechanisms of substrate selectivity used by this enzyme. Crystals of recombinant neurolysin have been grown in the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 157.8, b = 88.0, c = 58.4 A. Data have been collected to 2.3 A at 110 K with observed diffraction to 1.8 A. Circular dichroism measurements suggest that the enzyme is primarily alpha-helical, with little beta-strand secondary structure. Sequence-based secondary-structure prediction supports this conclusion.


Subject(s)
Metalloendopeptidases/chemistry , Animals , Circular Dichroism , Crystallization , Escherichia coli , Metalloendopeptidases/genetics , Metalloendopeptidases/metabolism , Protein Structure, Secondary , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Substrate Specificity , X-Ray Diffraction
11.
Brain Res Mol Brain Res ; 80(1): 88-98, 2000 Aug 14.
Article in English | MEDLINE | ID: mdl-11039732

ABSTRACT

REST4 is a neuron specific truncated form of the transcription factor REST/NRSE derived by alternative splicing. REST4 was previously shown to block the repressor activity of REST/NRSF by forming a hetero-oligomer, Shimojo et al. [Mol. Cell. Biol. 19 (1999) 6788-6795]. A series of deletion mutants have now been used to characterize REST4 in terms of its structure and DNA binding. REST4 was found to be O-glycosylated between between residues 87 and 152. Binding of REST4 to the cholinergic RE-1/NRSE was approximately 1/10 to 1/20 as strong as full length REST/NRSF. DNA binding was enhanced by deletion of the first 86 residues and was found to require all four of the C-terminal zinc fingers as well as a twelve amino acid sequence preceding the first of these zinc fingers. REST4 can form homo-oligomers, however only the monomer was found to bind to DNA. REST4 binds to the 3' sequence of the cholinergic NRSE suggesting an anti-parallel orientation of the protein to the DNA.


Subject(s)
Alternative Splicing , DNA-Binding Proteins/physiology , Repressor Proteins/physiology , Transcription Factors/physiology , Animals , Cells, Cultured , DNA/metabolism , DNA-Binding Proteins/genetics , Electrophoresis, Polyacrylamide Gel , Glycosylation , Humans , PC12 Cells , Polymers , Protein Binding , Protein Conformation , Rats , Repressor Proteins/genetics , Structure-Activity Relationship , Transcription Factors/genetics , Zinc Fingers/genetics
12.
Exp Neurol ; 165(2): 306-26, 2000 Oct.
Article in English | MEDLINE | ID: mdl-10993691

ABSTRACT

The cyto- and chemoarchitecture of basal forebrain cholinergic neurons (BFCN) was investigated in the lower primate, the common marmoset (Callithrix jacchus). A large population of magnocellular, hyperchromic, and choline acetyltransferase (ChAT)-positive neurons was detected in the marmoset basal forebrain. The distribution of these neurons was similar to those in higher primates. Thus, ChAT-positive neurons were observed in the medial septum (Ch2), the vertical (Ch2) and horizontal (Ch3) limbs of the diagonal band of Broca, and the nucleus basalis of Meynert (Ch4). The Ch4 complex was relatively well differentiated and displayed distinct sectors. We detected anterior (Ch4a, with a medial and a lateral subdivision), intermediate (Ch4i, with a dorsal and a ventral subdivision), and posterior (Ch4p) sectors in the marmoset Ch4. The Ch4i was relatively small while the Ch4p was large. Similar to the rodent, the marmoset Ch1 extended quite a distance posteriorly, and the Ch4p displayed a major interstitial component distributed within the globus pallidus, its medullary laminae, and the internal capsule. Virtually all of the marmoset BFCN displayed acetylcholinesterase activity, and low affinity (p75(NTR)) and high affinity (Trk) neurotrophin receptor immunoreactivity. A majority contained immunoreactivity for calbindin-D(28K) and calretinin. Many of the Ch4 neurons also displayed tyrosine hydroxylase immunoreactivity. The BFCN lacked galanin immunoreactivity, but were innervated by galanin-positive fibers. None of the marmoset BFCN were NADPH-d-positive. Thus, the BFCN display major anatomical and biochemical differences in the marmoset when compared with higher primates. The marmoset BFCN also display many characteristics common to other primates. This fact, combined with the relatively short life span of the marmoset, indicates that this species may be ideal for studies of age-related changes in the BFCN.


Subject(s)
Acetylcholinesterase/analysis , Callithrix/anatomy & histology , Choline O-Acetyltransferase/analysis , Cholinergic Fibers/chemistry , Prosencephalon/chemistry , Animals , Female , Male , Prosencephalon/cytology
13.
Eur J Pharmacol ; 397(2-3): 327-34, 2000 Jun 02.
Article in English | MEDLINE | ID: mdl-10844131

ABSTRACT

Alcohol consumption was investigated in mice which were rendered deficient in the peptide-degrading enzyme neutral endopeptidase (EC 3.4.24.11) (NEP-/-) by gene targeting and compared to alcohol consumption in corresponding wild type mice (NEP+/+). Mice were offered a free choice to drink tap water or 10% alcohol. The NEP-/- mice consumed significantly more alcohol ( approximately 42%) than the NEP+/+ mice, whereas no significant differences were observed in the total fluid consumption. The daily food consumption of alcohol naive NEP-/- animals was elevated ( approximately 29%). Furthermore, the activities of peptidases closely related to neutral endopeptidase were analysed ex vivo in several brain regions from NEP-/- and NEP+/+ mice not treated with alcohol. There was no obvious compensation for the total loss of neutral endopeptidase by the functionally related peptidases angiotensin-converting enzyme and aminopeptidase N. In vitro, the degradation of exogenously applied [Leu(5)]enkephalin was not reduced in membrane preparations of those brain regions assayed in NEP-/- mice. A small reduction in [Leu(5)]enkephalin degradation was detected in striatal membrane preparations of NEP-/- mice, if aminopeptidase N was additionally blocked by bestatin or amastatin.


Subject(s)
Alcohol Drinking , Neprilysin/metabolism , Animals , Auditory Cortex/metabolism , Brain/enzymology , CD13 Antigens/metabolism , Cerebral Cortex/metabolism , Corpus Striatum/metabolism , Enkephalin, Leucine/metabolism , Genotype , Hippocampus/metabolism , Male , Membranes/metabolism , Mesencephalon/metabolism , Mice , Mice, Knockout , Neprilysin/genetics , Olfactory Bulb/metabolism , Peptidyl-Dipeptidase A/metabolism , Thalamus/metabolism
14.
J Biol Chem ; 275(26): 19545-51, 2000 Jun 30.
Article in English | MEDLINE | ID: mdl-10764809

ABSTRACT

The subsite specificity of rat nardilysin was investigated using fluorogenic substrates of the type 2-aminobenzoyl-GGX(1)X(2)RKX(3)GQ-ethylenediamine-2,4- dinitrophenyl, where P(2), P(2)', and P(3) residues were varied. (The nomenclature of Schechter and Berger (Schechter, I., and Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162) is used where cleavage of a peptide occurs between the P(1) and P(1)' residues, and adjacent residues are designated P(2), P(3), P(2)', P(3)', etc.) There was little effect on K(m) among different residues at any of these positions. In contrast, residues at each position affected k(cat), with P(2) residues having the greatest effect. The S(3), S(2), and S(2)' subsites differed in their amino acid preference. Tryptophan and serine, which produced poor substrates at the P(2) position, were among the best P(2)' residues. The specificity at P(3) was generally opposite that of P(2). Residues at P(2), and to a lesser extent at P(3), influenced the cleavage site. At the P(2) position, His, Phe, Tyr, Asn, or Trp produced cleavage at the amino side of the first basic residue. In contrast, a P(2) Ile or Val produced cleavage between the dibasic pair. Other residues produced intermediate effects. The pH dependence for substrate binding showed that the enzyme prefers to bind a protonated histidine. A comparison of the effect of arginine or lysine at the P(1)' or P(1) position showed that there is a tendency to cleave on the amino side of arginine and that this cleavage produces the highest k(cat) values.


Subject(s)
Metalloendopeptidases/chemistry , Metalloendopeptidases/metabolism , Amino Acids/metabolism , Animals , Binding Sites , Chromatography, High Pressure Liquid , Fluorometry , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Peptides/metabolism , Rats , Substrate Specificity , Time Factors
15.
J Biol Chem ; 275(26): 19942-8, 2000 Jun 30.
Article in English | MEDLINE | ID: mdl-10748073

ABSTRACT

Metabolic labeling of a mutant PC12 cell line, A123.7, expressing recombinant rat vesicular acetylcholine transporter (VAChT) with radiolabeled inorganic phosphate was used to demonstrate phosphorylation of the transporter on a serine residue. Mutational analysis was used to demonstrate that serine 480, which is located on the COOH-terminal cytoplasmic tail, is the sole phosphorylation site. Phosphorylation of serine 480 was attributable to the action of protein kinase C. Using a permanently dephosphorylated form of rat VAChT, S480A rVAChT, it was shown that this mutant displays the same kinetics for the transport of acetylcholine and the binding of the inhibitor vesamicol as does the wild type transporter. However, sucrose gradient density centrifugation showed that, unlike wild type VAChT, the S480A mutant did not localize to synaptic vesicles. These results suggest that phosphorylation of serine 480 of VAChT is involved in the trafficking of this transporter.


Subject(s)
Carrier Proteins/metabolism , Membrane Transport Proteins , Vesicular Transport Proteins , Acetylcholine/metabolism , Acetylcholine/pharmacokinetics , Amino Acid Sequence , Animals , Binding Sites , Blotting, Western , Carrier Proteins/biosynthesis , Carrier Proteins/chemistry , Cell Nucleus/metabolism , Centrifugation, Density Gradient , Cytoplasm/metabolism , Immunoblotting , Molecular Sequence Data , Mutagenesis, Site-Directed , Neuromuscular Depolarizing Agents/pharmacokinetics , PC12 Cells , Phosphorylation , Piperidines/pharmacokinetics , Precipitin Tests , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Protein Structure, Tertiary , Rats , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Serine/metabolism , Synaptophysin/biosynthesis , Transfection , Vesicular Acetylcholine Transport Proteins
16.
J Biol Chem ; 275(9): 6175-80, 2000 Mar 03.
Article in English | MEDLINE | ID: mdl-10692409

ABSTRACT

The function of positively charged residues and the interaction of positively and negatively charged residues of the rat vesicular acetylcholine transporter (rVAChT) were studied. Changing Lys-131 in transmembrane domain helix 2 (TM2) to Ala or Leu eliminated transport activity, with no effect on vesamicol binding. However, replacement by His or Arg retained transport activity, suggesting a positive charge in this position is critical. Mutation of His-444 in TM12 or His-413 in the cytoplasmic loop between TM10 and TM11 was without effect on ACh transport, but vesamicol binding was reduced with His-413 mutants. Changing His-338 in TM8 to Ala or Lys did not effect ACh transport, however replacement with Cys or Arg abolished activity. Mutation of both of the transmembrane histidines or all three of the luminal loop histidines showed no change in acetylcholine transport. The mutant H338A/D398N between oppositely charged residues in transmembrane domains showed no vesamicol binding, however the charge reversal mutant H338D/D398H restored binding. This suggests that His-338 forms an ion pair with Asp-398. The charge neutralizing mutant K131A/D425N or the charge exchanged mutant K131D/D425K did not restore ACh transport. Taken together these results provide new insights into the tertiary structure in VAChT.


Subject(s)
Carrier Proteins/genetics , Membrane Transport Proteins , Vesicular Transport Proteins , Acetylcholine/metabolism , Animals , Biological Transport/genetics , Blotting, Western , Centrifugation, Density Gradient , Glycosylation , Histidine/genetics , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mutation , Piperidines/metabolism , Protein Binding/genetics , Protein Structure, Tertiary , Protons , Rats , Sequence Homology, Amino Acid , Static Electricity , Vesicular Acetylcholine Transport Proteins
17.
Mol Cell Endocrinol ; 170(1-2): 131-42, 2000 Dec 22.
Article in English | MEDLINE | ID: mdl-11162897

ABSTRACT

Transcription of the human neutral endopeptidase 24.11 (NEP) gene is androgen regulated in prostate cancer cells. Homology search identified a sequence GTCACAaagAGTTCT similar to the ARE consensus sequence GGTACAnnnTGTTCT within the 3'-untranslated region of the NEP mRNA. A double-stranded radiolabelled oligonucleotide containing this NEP-ARE sequence formed a DNA-protein complex with nuclear proteins from LNCaP cells or COS-7 cells co-transfected with an androgen receptor (AR) expression vector, and with full-length AR synthesized by baculovirus in mobility shift assays. Unlabeled NEP-ARE or consensus ARE but not mutated NEP-ARE replaced radiolabelled NEP-ARE. Steroid-dependent enhancement of transcription was assayed by transfecting ptkCAT reporter constructs containing the NEP-ARE into CV-1/AR cells and prostate cancer cells (PC-3/AR). Enhancement of chloramphenicol acetyltransferase (CAT) activity was increased four-fold by androgen, seven-fold by dexamethasone and three-fold by progesterone in CV-1/AR cells, and the NEP-ARE bound to glucocorticoid and progesterone receptor in mobility shift assays. We next performed DNase-I footprinting analysis of the NEP promoter and identified a 23 bp sequence GGTGCGGGTCGGAGGGATGCCCA (NEP-ARR) which was protected from DNase I cleavage by nuclear extracts from COS-7 cells expressing AR. This sequence was 62.5% homologous to an androgen responsive region (PSA-ARR) identified in the promoter of the prostate specific antigen (PSA) gene. A double-stranded radiolabelled oligonucleotide containing this NEP-ARR sequence formed DNA-protein complex with AR but not GR proteins. Unlabeled NEP-ARR, PSA-ARR and NEP-ARE replaced radiolabelled NEP-ARR. Steroid-dependent enhancement of transcription assays in PC-3/AR cells revealed that the enhancement of CAT activity was increased 2.3-fold by androgen, but not by glucocorticoid or progesterone. In a thymidine kinase promoter, the NEP-ARE and NEP-ARR together stimulated a five-fold increase in promoter activity in PC cells. These data suggest that steroid regulation of the NEP gene involves at least two elements including a typical ARE which binds androgen, progesterone and glucocorticoid receptors, and a unique ARR which only binds androgen receptor.


Subject(s)
Androgens/metabolism , Neprilysin/genetics , Response Elements/genetics , Androgens/pharmacology , Genes, Reporter , Humans , Male , Promoter Regions, Genetic/drug effects , Prostatic Neoplasms/pathology , Protein Binding , Receptors, Glucocorticoid/metabolism , Receptors, Progesterone/metabolism , Repetitive Sequences, Nucleic Acid , Transcription, Genetic/drug effects , Tumor Cells, Cultured
18.
J Mol Neurosci ; 15(3): 205-14, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11303784

ABSTRACT

The expression pattern of the repressor element-1 silencing transcription factor (REST) also known as the neuron-restrictive silencer factor (NRSF) and its truncated forms have been analyzed in the neuroblastoma cell lines, NS20Y and NIE115 and in NIH3T3 cells. The neuroblastoma cell lines express transcripts of REST/NRSF and its neuron-specific truncated form REST4; with REST4 being the major transcript. NIH3T3 cells express predominantly REST/NRSF, with no detectable REST4. The cellular localization of REST4, determined using a REST4-GFP fusion protein, was shown to be nuclear. Mutational analysis implicates the zinc finger domains as the nuclear-targeting signal. Analysis of reporter-gene activities in the NS20Y cell line showed that the presence of four RE-1/NRSE sequences did not affect promoter activity. However, coexpression of exogenous REST4 produces a small increase in promoter activity of the reporter plasmid, whereas expression of exogenous REST/NRSF leads to repression. In the NIH3T3 cell line, the RE-1/NRSE sequence leads to repression of reporter-gene activity, whereas introduction of exogenous REST4 leads to de-repression. These data indicate that REST4 does not act as a transcriptional repressor. However, they support a mechanism where REST4 can block the repressor activity of REST/NRSF.


Subject(s)
Cell Compartmentation/genetics , Cell Nucleus/genetics , Gene Expression Regulation, Neoplastic/physiology , Repressor Proteins/genetics , Transcription Factors/genetics , Tumor Cells, Cultured/metabolism , Acetylcholine/biosynthesis , Acetylcholine/genetics , Alternative Splicing/genetics , Animals , Brain/cytology , Brain/embryology , Brain/metabolism , Cell Nucleus/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Isomerism , Mice , Neuroblastoma , Neurons/cytology , Neurons/metabolism , Promoter Regions, Genetic/physiology , Protein Structure, Tertiary/genetics , Repressor Proteins/metabolism , Stem Cells/cytology , Stem Cells/metabolism , Transcription Factors/metabolism , Transcription, Genetic/physiology , Tumor Cells, Cultured/cytology
19.
Nat Immunol ; 1(5): 413-8, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11062501

ABSTRACT

The proteasome generates exact major histocompatibility complex (MHC) class I ligands as well as NH2-terminal-extended precursor peptides. The proteases responsible for the final NH2-terminal trimming of the precursor peptides had, until now, not been determined. By using specific selective criteria we purified two cytosolic proteolytic activities, puromycin-sensitive aminopeptidase and bleomycin hydrolase. These proteases could remove NH2-terminal amino acids from the vesicular stomatitis virus nucleoprotein cytotoxic T cell epitope 52-59 (RGYVYQGL) resulting, in combination with proteasomes, in the generation of the correct epitope. Our data provide evidence for the existence of redundant systems acting downstream of the proteasome in the antigen-processing pathway for MHC class I molecules.


Subject(s)
Endopeptidases/metabolism , Histocompatibility Antigens Class I/metabolism , Nucleocapsid Proteins , Amino Acid Chloromethyl Ketones/pharmacology , Amino Acid Sequence , Aminopeptidases/isolation & purification , Aminopeptidases/metabolism , Animals , Antigens, Viral/chemistry , Antigens, Viral/genetics , Antigens, Viral/metabolism , Cell Line , Cysteine Endopeptidases/isolation & purification , Cysteine Endopeptidases/metabolism , Endopeptidases/isolation & purification , Epitopes/chemistry , Epitopes/genetics , Epitopes/metabolism , Humans , Ligands , Mice , Molecular Sequence Data , Multienzyme Complexes/metabolism , Nucleocapsid/genetics , Nucleocapsid/immunology , Nucleocapsid/metabolism , Proteasome Endopeptidase Complex , Protein Processing, Post-Translational/drug effects , Vesicular stomatitis Indiana virus/genetics , Vesicular stomatitis Indiana virus/immunology
20.
Neuroscience ; 94(1): 21-31, 1999.
Article in English | MEDLINE | ID: mdl-10613493

ABSTRACT

Neurochemical and functional abnormalities of the striatum have been reported in schizophrenic brains, but the cellular substrates of these changes are not known. We hypothesized that schizophrenia may involve an abnormality in one of the key modulators of striatal output, the cholinergic interneuron. We measured the densities of cholinergic neurons in the striatum in schizophrenic and control brains in a blind analysis, using as a marker of this cell population immunoreactivity for choline acetyltransferase, the synthetic enzyme of acetylcholine. As an independent marker, we used immunoreactivity for calretinin, a protein which is co-localized with choline acetyltransferase in virtually all of the cholinergic interneurons of the striatum. A significant decrease in choline acetyltransferase-positive and calretinin-positive cell densities was found in the schizophrenic cases compared with controls in the striatum as a whole [for the choline acetyltransferase-positive cells: controls: 3.21 +/- 0.48 cells/mm2 (mean +/- S.D.), schizophrenics: 2.43 +/- 0.68 cells(mm2; P < 0.02]. The decrease was patchy in nature and most prominent in the ventral striatum (for the choline acetyltransferase-positive cells: controls: 3.47 +/- 0.59 cells/mm2, schizophrenics: 2.52 +/- 0.64 cells/ mm2; P < 0.005) which included the ventral caudate nucleus and nucleus accumbens region. Three of the schizophrenic cases with the lowest densities of cholinergic neurons had not been treated with neuroleptics for periods from more than a month to more than 20 years. A decrease in the number or function of the cholinergic interneurons of the striatum may disrupt activity in the ventral striatal-pallidal-thalamic-prefrontal cortex pathway and thereby contribute to abnormalities in function of the prefrontal cortex in schizophrenia.


Subject(s)
Cholinergic Fibers/pathology , Corpus Striatum/pathology , Interneurons/pathology , Schizophrenia/metabolism , Schizophrenia/pathology , Adult , Aged , Aged, 80 and over , Brain Chemistry , Calbindin 2 , Cell Count , Choline O-Acetyltransferase/analysis , Cholinergic Fibers/chemistry , Cholinergic Fibers/enzymology , Corpus Striatum/metabolism , Humans , Interneurons/enzymology , Interneurons/ultrastructure , Middle Aged , S100 Calcium Binding Protein G/analysis
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