RESUMO
BACKGROUND: Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), is generally recognized as a diagnostic and therapeutic cancer antigen and a molecular address for targeted imaging and drug delivery studies. Due to its significance in cancer research, numerous monoclonal antibodies (mAbs) against GCPII have been described and marketed in the past decades. Unfortunately, some of these mAbs are poorly characterized, which might lead to their inappropriate use and misinterpretation of the acquired results. METHODS: We collected the 13 most frequently used mAbs against GCPII and quantitatively characterized their binding to GCPII by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Using a peptide library, we mapped epitopes recognized by a given mAb. Finally, we assessed the applicability of these mAbs to routine experimental setups, including Western blotting, immunohistochemistry, and flow cytometry. RESULTS: ELISA and SPR analyses revealed that mAbs J591, J415, D2B, 107-1A4, GCP-05, and 2G7 bind preferentially to GCPII in native form, while mAbs YPSMA-1, YPSMA-2, GCP-02, GCP-04, and 3E6 bind solely to denatured GCPII. mAbs 24.4E6 and 7E11-C5.3 recognize both forms of GCPII. Additionally, we determined that GCP-02 and 3E6 cross-react with mouse GCPII, while GCP-04 recognizes GCPII and GCPIII proteins from both human and mouse. CONCLUSION: This comparative analysis provides the first detailed quantitative characterization of the most commonly used mAbs against GCPII and can serve as a guideline for the scientific community to use them in a proper and efficient way.
Assuntos
Adenocarcinoma/imunologia , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Antígeno Prostático Específico/imunologia , Neoplasias da Próstata/imunologia , Adenocarcinoma/diagnóstico , Adenocarcinoma/metabolismo , Western Blotting , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Masculino , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/metabolismoRESUMO
Large collections of protein-encoding open reading frames (ORFs) established in a versatile recombination-based cloning system have been instrumental to study protein functions in high-throughput assays. Such 'ORFeome' resources have been developed for several organisms but in virology, plasmid collections covering a significant fraction of the virosphere are still needed. In this perspective, we present ViralORFeome 1.0 (http://www.viralorfeome.com), an open-access database and management system that provides an integrated set of bioinformatic tools to clone viral ORFs in the Gateway(R) system. ViralORFeome provides a convenient interface to navigate through virus genome sequences, to design ORF-specific cloning primers, to validate the sequence of generated constructs and to browse established collections of virus ORFs. Most importantly, ViralORFeome has been designed to manage all possible variants or mutants of a given ORF so that the cloning procedure can be applied to any emerging virus strain. A subset of plasmid constructs generated with ViralORFeome platform has been tested with success for heterologous protein expression in different expression systems at proteome scale. ViralORFeome should provide our community with a framework to establish a large collection of virus ORF clones, an instrumental resource to determine functions, activities and binding partners of viral proteins.
Assuntos
Biologia Computacional/métodos , Bases de Dados Genéticas , Bases de Dados de Ácidos Nucleicos , Bases de Dados de Proteínas , Genes Virais , Fases de Leitura Aberta , Clonagem Molecular , Biologia Computacional/tendências , Técnicas Genéticas , Genoma Viral , Armazenamento e Recuperação da Informação/métodos , Internet , Estrutura Terciária de Proteína , Software , Interface Usuário-ComputadorRESUMO
Innate immunity has evolved complex molecular pathways to protect organisms from viral infections. One pivotal line of cellular defense is the induction of the antiviral effect of interferon. To circumvent this primary response and achieve their own replication, viruses have developed complex molecular strategies. Here, we provide a systems-level study of the human type I interferon system subversion by the viral proteome, by reconstructing the underlying protein-protein interaction network. At this network level, viruses establish a massive and a gradual attack, from receptors to transcription factors, by interacting preferentially with highly connected and central proteins as well as interferon-induced proteins. We also demonstrate that viruses significantly target 22% of the proteins directly interacting with the type I interferon system network, suggesting the relevance of our network-based method to identify new candidates involved in the regulation of the antiviral response. Finally, based on the comparative analysis of interactome profiles across four viral families, we provide evidence of common and differential targeting strategies.
Assuntos
Interações Hospedeiro-Patógeno/imunologia , Interferon Tipo I/imunologia , Mapeamento de Interação de Proteínas/métodos , Biologia de Sistemas/métodos , Vírus/imunologia , Bases de Dados Genéticas , Flaviviridae/imunologia , Herpesviridae/imunologia , Humanos , Papillomaviridae/imunologia , Retroviridae/imunologia , Transdução de Sinais , Estatísticas não ParamétricasRESUMO
A proteome-wide mapping of interactions between hepatitis C virus (HCV) and human proteins was performed to provide a comprehensive view of the cellular infection. A total of 314 protein-protein interactions between HCV and human proteins was identified by yeast two-hybrid and 170 by literature mining. Integration of this data set into a reconstructed human interactome showed that cellular proteins interacting with HCV are enriched in highly central and interconnected proteins. A global analysis on the basis of functional annotation highlighted the enrichment of cellular pathways targeted by HCV. A network of proteins associated with frequent clinical disorders of chronically infected patients was constructed by connecting the insulin, Jak/STAT and TGFbeta pathways with cellular proteins targeted by HCV. CORE protein appeared as a major perturbator of this network. Focal adhesion was identified as a new function affected by HCV, mainly by NS3 and NS5A proteins.
Assuntos
Hepatite C/metabolismo , Proteínas Virais/metabolismo , Hepacivirus/metabolismo , Hepacivirus/fisiologia , Humanos , Ligação Proteica , Técnicas do Sistema de Duplo-HíbridoRESUMO
Glutamate carboxypeptidase II (GCPII) and its splice variants, paralogs and human homologs represent a family of proteins with diverse tissue distribution, cellular localization and largely unknown function which have been explored only recently. While GCPII itself has been thoroughly studied from different perspectives, as clearly documented in this series of reviews, very little is known about other members of its family, even though they might be biologically relevant. Differential expression of individual GCPII splice variants is associated with tumor progression and prognosis of prostate cancer. The best studied GCPII homolog, GCPIII or NAALADase II, may be a valid pharmaceutical target for itself since it may compensate for a lack of normal GCPII enzymatic activity. Detailed molecular characterization of this family of proteins is thus very important not only with respect to the potential therapeutic use of GCPII inhibitors, but also for better understanding of the biological role of GCPII within as well as outside the nervous system.