Z lymfocytů pacientů s AIDS léčených inhibitory proteasy HIV budou sekvenovány a charakterisovány in vitro i ve tkáňové kultuře mutanty proteasy, resistentní vůči původnímu inhibitoru, a otestovány pomocí panelu struktuálně rozdílných inhibitorů.; Resistant mutants of the HIV protease from the blood lymphocytes of AIDS patients treated with protease inhibitors will be amplified, sequenced, characterized in vitro and in tissue culture and tested with a panel of structurally different inhibitors.

• MeSH
• AIDS terapie   MeSH
• antibiotická rezistence MeSH
• molekulární modely MeSH
• molekulární struktura MeSH

• Konspekt
• Virologie
• NLK Obory
• dermatovenerologie
• biologie
• virologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

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.

• MeSH
• alanin metabolismus   MeSH
• biologické modely MeSH
• dipeptidy genetika   metabolismus   MeSH
• financování organizované MeSH
• glutamátkarboxypeptidasa II genetika   chemie   metabolismus   MeSH
• hydrolýza MeSH
• kinetika MeSH
• krystalografie rentgenová MeSH
• kvantová teorie MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutageneze MeSH
• substituce aminokyselin MeSH
• substrátová specifita genetika   MeSH
• termodynamika MeSH
• vazba proteinů genetika   MeSH
• vazebná místa genetika   MeSH
• vodíková vazba MeSH
• výpočetní biologie metody   MeSH
• zinek chemie   MeSH
• Check Tag
• lidé MeSH

BACKGROUND: Prostate specific membrane antigen (PSMA) is a type II transmembrane protein overexpressed in prostate cancer as well as in the neovasculature of several non-prostatic solid tumors. In addition to full-length PSMA, several splice variants exist in prostatic tissue. Notably, the N-terminally truncated PSMA variant, termed PSM', is prevalent in healthy prostate, and the ratio of PSMA/PSM' mRNA has been shown to correlate with cancer progression. The widely accepted hypothesis is that the PSM' protein is a translation product arising from the alternatively spliced PSM' mRNA. METHODS: Differential ultracentrifugation, cell surface biotinylation, Western blotting, and enzyme activity measurement were used to study the origin and localization of the PSMA/PSM' variants in prostatic (LNCaP; lymph-node carcinoma of the prostate) and non-prostatic (HEK293) cell lines. These experiments were further complemented by analysis of the N-glycosylation patterns of the PSMA/PSM' proteins and by site-directed mutagenesis. RESULTS: We identified PSM' protein expression in both the LNCaP cell line and a non-cancerous HEK293 human cell line transfected with a plasmid encoding full-length PSMA. Differential centrifugation revealed that PSM' is localized predominantly to the cytosol of both these cell lines and is proteolytically active. Furthermore, the PSM' protein is N-glycosylated by a mixture of high-mannose and complex type oligosaccharides and therefore trafficked beyond the cis-Golgi compartment. CONCLUSIONS: Our data suggest that the PSM' protein is likely not generated by alternative splicing of the PSMA gene but by different mechanism, probably via an endoproteolytic cleavage of the full-length PSMA.

• MeSH
• adenokarcinom metabolismus   patologie   MeSH
• buněčné linie MeSH
• financování organizované MeSH
• glykosylace MeSH
• ledviny cytologie   embryologie   metabolismus   MeSH
• lidé MeSH
• lyzozomy metabolismus   MeSH
• mikrozomy metabolismus   MeSH
• místa sestřihu RNA genetika   MeSH
• mitochondrie metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádory prostaty metabolismus   patologie   MeSH
• prostatický specifický antigen genetika   chemie   metabolismus   MeSH
• transfekce MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH

BACKGROUND: Prostate specific membrane antigen (PSMA), also called glutamate carboxypeptidase II (GCPII), is a target enzyme for diagnosis and treatment of prostate cancer. Moreover, it is upregulated in the vasculature of most solid tumors and is therefore a potential target for the generation of novel antineoplastics. In this context, we analyze the possibility of using rat and pig as animal models for enzymologic and in vivo studies. METHODS: We prepared the recombinant extracellular part of human, rat, and pig GCPII in S2 cell media and characterized the activity and inhibition profiles of the three orthologs by radioenzymatic assay. We performed Western blot analysis of GCPII expression in human, rat, and pig tissues using the monoclonal antibody GCP-04 and confirmed these findings by activity measurements and immunohistochemistry. RESULTS: The three recombinant proteins show similar specific enzymatic activities and inhibition profiles. Tissue expression analysis revealed that most of the pig and human tissues show at least some GCPII-positivity, while the expression pattern in rat is more restricted. Moreover, tissues such as prostate and testes exhibit different GCPII expression levels among the species studied. CONCLUSIONS: The rat and pig orthologs of GCPII seem to be suitable to approximate human GCPII in enzymologic studies. However, the diffuse expression pattern of GCPII in animal and human tissues could be a caveat for the potential utilization of GCPII-targeted anticancer drugs. Furthermore, variations in GCPII tissue distribution among the species studied should be considered when using rat or pig as models for antineoplastic drug discovery.

• MeSH
• druhová specificita MeSH
• financování organizované MeSH
• krysa rodu rattus MeSH
• ledviny enzymologie   patologie   MeSH
• lidé MeSH
• mícha enzymologie   patologie   MeSH
• miniaturní prasata MeSH
• modely u zvířat MeSH
• molekulární sekvence - údaje MeSH
• potkani inbrední LEW MeSH
• prasata MeSH
• prostata enzymologie   patologie   MeSH
• prostatický specifický antigen analýza   genetika   metabolismus   MeSH
• regulace genové exprese enzymů MeSH
• sekvence aminokyselin MeSH
• testis enzymologie   patologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• srovnávací studie MeSH

Glutamate carboxypeptidase II (GCPII) is a transmembrane glycoprotein expressed in various tissues. When expressed in the brain it cleaves the neurotransmitter N-acetylaspartylglutamate (NAAG), yielding free glutamate. In jejunum it hydrolyzes folylpoly-gamma-glutamate, thus facilitating folate absorption. The prostate form of GCPII, known as prostate specific membrane antigen (PSMA), is an established cancer marker. The NAAG-hydrolyzing activity of GCPII has been implicated in a number of pathological conditions in which glutamate is neurotoxic (e.g. amyotrophic lateral sclerosis, Huntington's disease, Alzheimer's disease, epilepsy, schizophrenia, and stroke). Inhibition of GCPII was shown to be neuroprotective in tissue culture and in animal models. GCPII is therefore an interesting putative therapeutic target. However, only very limited and controversial data on the expression and localization of GCPII in human brain are available. Therefore, we set out to analyze the activity and expression of GCPII in various compartments of the human brain using a radiolabeled substrate of the enzyme and the novel monoclonal antibody GCP-04, which recognizes an epitope on the extracellular portion of the enzyme and is more sensitive to GCPII than to the homologous GCPIII. We show that this antibody is more sensitive in immunoblots than the widely used antibody 7E11. By Western blot, we show that there are approximately 50-300 ng of GCPII/mg of total protein in human brain, depending on the specific area. Immunohistochemical analysis revealed that astrocytes specifically express GCPII in all parts of the brain. GCPII is enzymatically active and the level of activity follows the expression pattern. Using pure recombinant GCPII and homologous GCPIII, we conclude that GCPII is responsible for the majority of overall NAAG-hydrolyzing activity in the human brain.

• MeSH
• aktivace enzymů genetika   MeSH
• antigeny povrchové analýza   imunologie   metabolismus   MeSH
• astrocyty enzymologie   MeSH
• dipeptidy metabolismus   MeSH
• financování organizované MeSH
• glutamátkarboxypeptidasa II analýza   imunologie   metabolismus   MeSH
• imunohistochemie metody   MeSH
• kyselina glutamová biosyntéza   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mapování epitopu metody   MeSH
• molekulární modely MeSH
• mozek anatomie a histologie   enzymologie   MeSH
• protilátky imunologie   MeSH
• radioligandová zkouška metody   MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• terciární struktura proteinů genetika   MeSH
• western blotting MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH

Human glutamate carboxypeptidase II [GCPII (EC 3.4.17.21)] is recognized as a promising pharmacological target for the treatment and imaging of various pathologies, including neurological disorders and prostate cancer. Recently reported crystal structures of GCPII provide structural insight into the organization of the substrate binding cavity and highlight residues implicated in substrate/inhibitor binding in the S1' site of the enzyme. To complement and extend the structural studies, we constructed a model of GCPII in complex with its substrate, N-acetyl-l-aspartyl-l-glutamate, which enabled us to predict additional amino acid residues interacting with the bound substrate, and used site-directed mutagenesis to assess the contribution of individual residues for substrate/inhibitor binding and enzymatic activity of GCPII. We prepared and characterized 12 GCPII mutants targeting the amino acids in the vicinity of substrate/inhibitor binding pockets. The experimental results, together with the molecular modeling, suggest that the amino acid residues delineating the S1' pocket of the enzyme (namely Arg210) contribute primarily to the high affinity binding of GCPII substrates/inhibitors, whereas the residues forming the S1 pocket might be more important for the 'fine-tuning' of GCPII substrate specificity.

• MeSH
• antigeny povrchové genetika   chemie   metabolismus   MeSH
• financování organizované MeSH
• glutamátkarboxypeptidasa II antagonisté a inhibitory   genetika   chemie   metabolismus   MeSH
• kinetika MeSH
• krysa rodu rattus MeSH
• kyselina glutamová metabolismus   MeSH
• lidé MeSH
• molekulární modely MeSH
• mutageneze cílená MeSH
• myši MeSH
• sekvenční seřazení MeSH
• substrátová specifita MeSH
• vazebná místa MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH

Human glutamate carboxypeptidase II (GCPII) is a transmembrane metallopeptidase found mainly in the brain, small intestine, and prostate. In the brain, it cleaves N-acetyl-L-aspartyl-glutamate, liberating free glutamate. Inhibition of GCPII has been shown to be neuroprotective in models of stroke and other neurodegenerations. In prostate, it is known as prostate-specific membrane antigen, a cancer marker. Recently, human glutamate carboxypeptidase III (GCPIII), a GCPII homolog with 67% amino acid identity, was cloned. While GCPII is recognized as an important pharmaceutical target, no biochemical study of human GCPIII is available at present. Here, we report the cloning, expression, and characterization of recombinant human GCPIII. We show that GCPIII lacks dipeptidylpeptidase IV-like activity, its activity is dependent on N-glycosylation, and it is effectively inhibited by several known inhibitors of GCPII. In comparison to GCPII, GCPIII has lower N-acetyl-L-aspartyl-glutamate-hydrolyzing activity, different pH and salt concentration dependence, and distinct substrate specificity, indicating that these homologs might play different biological roles. Based on a molecular model, we provide interpretation of the distinct substrate specificity of both enzymes, and examine the amino acid residues responsible for the differences by site-directed mutagenesis. These results may help to design potent and selective inhibitors of both enzymes.

• MeSH
• antigeny povrchové analýza   genetika   metabolismus   MeSH
• financování organizované MeSH
• glutamátkarboxypeptidasa II analýza   genetika   chemická syntéza   metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• karboxypeptidasy analýza   genetika   chemická syntéza   metabolismus   MeSH
• klonování DNA metody   MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutageneze cílená MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH

• MeSH
• HIV infekce farmakoterapie   MeSH
• HIV-1 účinky léků   MeSH
• lidé MeSH
• oligopeptidy farmakologie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH