Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:Q04609 (prostate-specific membrane antigen)
 1,287 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane peptidase expressed in a number of tissues such as kidney, prostate and brain. The brain form of GCPII (also known as NAALADase) cleaves N-acetyl-aspartyl glutamate to yield free glutamate. Animal model experiments show that inhibition of GCPII prevents neuronal cell death during experimental ischaemia. GCPII thus represents an important target for the treatment of neuronal damage caused by excess glutamate. In this paper we report expression of an extracellular portion of human glutamate carboxypeptidase II (amino acids 44-750) in Drosophila Schneider's cells and its purification to homogeneity. A novel assay for hydrolytic activity of recombinant human GCPII (rhGCPII), based on fluorimetric detection of released alpha-amino groups was established, and used for its enzymological characterization. rhGCPII does not show dipeptidylpeptidase IV-like activity assigned to the native form of the enzyme previously. Using a complete set of protected dipeptides, substrate specificity of rhGCPII was elucidated. In addition to the previously described substrates, four novel compounds, Ac-Glu-Met, Ac-Asp-Met and, surprisingly, Ac-Ala-Glu and Ac-Ala-Met were identified as substrates for GCPII, and their respective kinetic constants determined. The glycosylation of rhGCPII was found indispensable for the enzymatic activity.
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PMID:Substrate specificity, inhibition and enzymological analysis of recombinant human glutamate carboxypeptidase II. 1190 94

Glutamate carboxypeptidase II (GCPII, NAALADase, or NAAG peptidase) is a catalytic zinc metallopeptidase. Its extracellular domain hydrolyzes the abundant neuropeptide, N-acetyl-L-aspartyl-L-glutamate (NAAG), to produce N-acetylaspartate and glutamate following the synaptic release of this transmitter. Thus, GCPII influences the extracellular concentrations of both glutamate and NAAG. NAAG activates group II metabotropic glutamate receptors, and activation of this receptor has been found to protect against anoxia-induced excitotoxic nerve cell death. In contrast, high levels of glutamate can be neurotoxic. Thus, GCPII is a potential therapeutic target for the reduction of excitotoxic levels of glutamate and enhancement of extracellular NAAG. To explore the structural basis of the interaction between GCPII and its inhibitors, we modeled the three-dimensional structure of the GCPII extracellular domain using a homology modeling approach. On the basis of the GCPII model, the structures of GCPII in complex with its potent inhibitors 2-(phosphonomethyl)pentanedioic acid (PMPA) and 4,4'-phosphinicobis(butane-1,3-dicarboxylic acid) (PBDA) were built by a computational docking method. The model of GCPII mainly consists of two alpha/beta/alpha sandwiches, between which two zinc ions are quadrivalently coordinated by the His379-Asp389-Asp455-H(2)O and the Asp389-Glu427-His555-H(2)O clusters, respectively. The ligand binding pocket is situated between these two sandwiches and is comprised of two subpockets: one is a surface-exposed highly positively charged subpocket; the other is a buried hydrophobic subpocket. The positively charged subpocket can accommodate the pharmacophore groups of inhibitor molecules (PMPA and PBDA) through the coordination of Zn(2+) with their phosphorus functionality and hydrogen-bonding interactions with Arg536, Arg538, and Ser456 (or Asn521), while the hydrophobic subpocket is engaged in hydrophobic and hydrogen-bonding interactions with the nonpharmacophore groups of PBDA. The predicted binding mode is consistent with the experimental data obtained from site-directed mutagenesis. On the basis of the predicted interaction mode, our structure-based design has led to a series of highly potent GCPII inhibitors.
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PMID:Molecular modeling of the interactions of glutamate carboxypeptidase II with its potent NAAG-based inhibitors. 1221 57

Glutamate carboxypeptidase II (GCPII, EC 3.14.17.21) is a membrane-bound enzyme found on the extracellular face ofglia. The gene for this enzyme is designated FOLH1 in humans and Folh1 in mice. This enzyme has been proposed to be responsible for inactivation of the neurotransmitter N-acetylaspartylglutamate (NAAG) following synaptic release. Mice harboring a disruption of the gene for GCPII/Folh1 were generated by inserting into the genome a targeting cassette in which the intron-exon boundary sequences of exons 1 and 2 were removed and stop codons were inserted in exons 1 and 2. Messenger RNA for GCPII was not detected by northern blotting or RT-PCR analysis of RNA from the brains of -/- mutant mice nor was GCPII protein detected on western blots of this tissue. These GCPII null mutant mice developed normally to adulthood and exhibited a normal range of neurologic responses and behaviors including mating, open field activity and retention of position in rotorod tests. No significant differences were observed among responses of wild type, heterozygous mutant and homozygous mutant mice on tail flick and hot plate latency tests. Glutamate, NAAG and mRNA for metabotropic glutamate receptor type 3 levels were not significantly altered in response to the deletion of glutamate carboxypeptidase II. A novel membrane-bound NAAG peptidase activity was discovered in brain, spinal cord and kidney of the GCPII knock out mice. The kinetic values for brain NAAG peptidase activity in the wild type and GCPII nullmutant were Vmax = 45 and 3 pmol/mg/min and Km = 2650 nm and 2494 nm, respectively. With the exception of magnesium and copper, this novel peptidase activity had a similar requirement for metal ions as GCPII. Two potent inhibitors of GCPII, 4,4'-phosphinicobis-(butane-1,3 dicarboxilic acid) (FN6) and 2-(phosphonomethyl)pentanedioic acid (2-PMPA) inhibited the residual activity. The IC50 value for 2-PMPA was about 1 nm for wild-type brain membrane NAAG peptidase activity consistent with its activity against cloned ratand human GCPII, and 88 nm for the activity in brain membranes of the null mutants.
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PMID:Deletion of the glutamate carboxypeptidase II gene in mice reveals a second enzyme activity that hydrolyzes N-acetylaspartylglutamate. 1235 25

Glutamate carboxypeptidase II (GCPII or prostate-specific membrane antigen or NAALADase) is an enzyme that catalyzes the hydrolysis of the neuropeptide N-acetylaspartylglutamate (NAAG) to N-acetylaspartate (NAA) and glutamate (G). Inhibitors of GCPII provide neuroprotection in a variety of animal models of central nervous system disorders. Neuroprotection is probably the result of increased NAAG concentrations and decreased levels of excess toxic glutamate. Consequently, GCPII inhibitors could be useful therapeutic agents where increased glutamate levels are the result of increased GCPII activity. Current GCPII in vitro activity assays are cumbersome or have limited sensitivity. In this report we describe a microplate assay to study GCPII inhibition that is most sensitive, efficient, and generates little waste. GCPII turnover number (k(cat)) was 4s(-1) and the binding constant (K(m)) for NAAG and GCPII was 130nM. The apparent association rate constant for GCPII and NAAG (k(cat)/K(m)) was 3 x 10(7)M(-1)s(-1). Inhibition studies with the GCPII inhibitor 2-(phosphonomethyl)pentanedioic acid (2-PMPA) demonstrated competitive inhibition with a K(i)=0.2nM.
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PMID:Kinetics and inhibition of glutamate carboxypeptidase II using a microplate assay. 1241 72

Glutamate carboxypeptidase II (GCPII) is a membrane peptidase expressed in the prostate, central and peripheral nervous system, kidney, small intestine, and tumor-associated neovasculature. The GCPII form expressed in the central nervous system, termed NAALADase, is responsible for the cleavage of N-acetyl-L-aspartyl-L-glutamate (NAAG) yielding free glutamate in the synaptic cleft, and is implicated in various pathologic conditions associated with glutamate excitotoxicity. The prostate form of GCPII, termed prostate-specific membrane antigen (PSMA), is up-regulated in cancer and used as an effective prostate cancer marker. Little is known about the structure of this important pharmaceutical target. As a type II membrane protein, GCPII is heavily glycosylated. In this paper we show that N-glycosylation is vital for proper folding and subsequent secretion of human GCPII. Analysis of the predicted N-glycosylation sites also provides evidence that these sites are critical for GCPII carboxypeptidase activity. We confirm that all predicted N-glycosylation sites are occupied by an oligosaccharide moiety and show that glycosylation at sites distant from the putative catalytic domain is critical for the NAAG-hydrolyzing activity of GCPII calling the validity of previously described structural models of GCPII into question.
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PMID:Identification of the N-glycosylation sites on glutamate carboxypeptidase II necessary for proteolytic activity. 1515 93

Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a zinc-dependent exopeptidase and an important therapeutic target for neurodegeneration and prostate cancer. The hydrolysis of N-acetyl-l-aspartyl-l-glutamate (N-Ac-Asp-Glu), the natural dipeptidic substrate of the GCPII, is intimately involved in cellular signaling within the mammalian nervous system, but the exact mechanism of this reaction has not yet been determined. To investigate peptide hydrolysis by GCPII in detail, we constructed a mutant of human GCPII [GCPII(E424A)], in which Glu424, a putative proton shuttle residue, is substituted with alanine. Kinetic analysis of GCPII(E424A) using N-Ac-Asp-Glu as substrate revealed a complete loss of catalytic activity, suggesting the direct involvement of Glu424 in peptide hydrolysis. Additionally, we determined the crystal structure of GCPII(E424A) in complex with N-Ac-Asp-Glu at 1.70 A resolution. The presence of the intact substrate in the GCPII(E424A) binding cavity substantiates our kinetic data and allows a detailed analysis of GCPII/N-Ac-Asp-Glu interactions. The experimental data are complemented by the combined quantum mechanics/molecular mechanics calculations (QM/MM) which enabled us to characterize the transition states, including the associated reaction barriers, and provided detailed information concerning the GCPII reaction mechanism. The best estimate of the reaction barrier was calculated to be DeltaG(++) approximately 22(+/-5) kcal x mol(-1), which is in a good agreement with the experimentally observed reaction rate constant (k(cat) approximately 1 s(-1)). Combined together, our results provide a detailed and consistent picture of the reaction mechanism of this highly interesting enzyme at the atomic level.
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PMID:Reaction mechanism of glutamate carboxypeptidase II revealed by mutagenesis, X-ray crystallography, and computational methods. 1930 71

Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a zinc metallopeptidase that hydrolyzes N-acetylaspartylglutamate (NAAG) into N-acetylaspartate (NAA) and glutamate in the nervous system. Inhibition of GCPII has the potential to reduce extracellular glutamate and represents an opportune target for treating neurological disorders in which excess glutamate is considered pathogenic. Furthermore, GCPII was found to be identical to a tumor marker, prostate-specific membrane antigen (PSMA), and has drawn significant interest as a diagnostic and/or therapeutic target in oncology. Over the past 15 years, tremendous efforts have been made in the discovery of potent GCPII inhibitors, particularly those with phosphorus-, urea- and thiol-based zinc binding groups. In addition, significant progress has been made in understanding the three-dimensional structural characteristics of GCPII in complex with various ligands. The purpose of this review article is to analyze the structure-activity relationships (SAR) of GCPII inhibitors reported to date, which are classified on the basis of their zinc-binding group. SAR and crystallographic data are evaluated in detail for each of these series to highlight the future challenges and opportunities to identify clinically viable GCPII inhibitors.
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PMID:Structure-activity relationships of glutamate carboxypeptidase II (GCPII) inhibitors. 2230 17

Glutamate carboxypeptidase II (GCPII) in the central nervous system is referred to as the prostate-specific membrane antigen (PSMA) in the periphery. PSMA serves as a target for imaging and treatment of prostate cancer and because of its expression in solid tumor neovasculature has the potential to be used in this regard for other malignancies as well. An overview of GCPII/PSMA in cancer, as well as a discussion of imaging and therapy of prostate cancer using a wide variety of PSMA-targeting agents is provided.
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PMID:GCPII imaging and cancer. 2230 13