RESUMO
Cancer/testis (CT) antigens are considered promising target molecules for immunotherapy. To efficiently identify potential CT antigens, a testis cDNA library was immunoscreened with sera from hepatocellular carcinoma (HCC) patients. We isolated 3 different antigens, AKAP3, CTp11, and UBQLN3. Although AKAP3 and CTp11 have been previously reported as CT antigens, this is the first time that these 2 antigens have been isolated from HCC patients by SEREX. Conventional RT-PCR analysis showed that AKAP3 was frequently present in HCC cell lines (5/7) and HCC tissues (5/10), and the gene was broadly expressed in several cancer types, including breast cancer cell lines (3/6), breast cancer tissues (6/9), colon cancer cell lines (3/10), colon cancer tissues (5/6), ovary cancer cell lines (6/8), ovary cancer tissues (11/16), lung cancer cell lines (4/7) and lung cancer tissues (6/13). By phage plaque analysis, anti-AKAP3 antibody was detected in sera from 15 of 27 HCC patients and 8 of 27 healthy donors. These data suggest that AKAP3 may be useful for diagnosis and immunotherapy in HCC patients.
Assuntos
Proteínas de Ancoragem à Quinase A/imunologia , Antígenos de Neoplasias/sangue , Carcinoma Hepatocelular/imunologia , Proteínas de Ligação a DNA/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias/imunologia , Ubiquitinas/imunologia , Proteínas de Ancoragem à Quinase A/sangue , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/isolamento & purificação , Idoso , Antígenos de Neoplasias/imunologia , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/terapia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/sangue , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/terapia , Masculino , Proteínas de Neoplasias/sangue , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/isolamento & purificação , Neoplasias/metabolismo , RNA Mensageiro/genética , Ubiquitinas/sangue , Ubiquitinas/genética , Ubiquitinas/isolamento & purificaçãoRESUMO
MOTIVATION: Favorable interaction between the regulatory subunit of the cAMP-dependent protein kinase (PKA) and a peptide in A-kinase anchoring proteins (AKAPs) is critical for translocating PKA to the subcellular sites where the enzyme phosphorylates its substrates. It is very hard to identify AKAPs peptides binding to PKA due to the high sequence diversity of AKAPs. RESULTS: We propose a hierarchical and efficient approach, which combines molecular dynamics (MD) simulations, free energy calculations, virtual mutagenesis (VM) and bioinformatics analyses, to predict peptides binding to the PKA RIIα regulatory subunit in the human proteome systematically. Our approach successfully retrieved 15 out of 18 documented RIIα-binding peptides. Literature curation supported that many newly predicted peptides might be true AKAPs. Here, we present the first systematic search for AKAP peptides in the human proteome, which is useful to further experimental identification of AKAPs and functional analysis of their biological roles.
Assuntos
Proteínas de Ancoragem à Quinase A/isolamento & purificação , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Peptídeos/isolamento & purificação , Proteômica/métodos , Proteínas de Ancoragem à Quinase A/química , Proteínas de Ancoragem à Quinase A/metabolismo , Sequência de Aminoácidos , Proteínas Quinases Dependentes de AMP Cíclico/química , Humanos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Subunidades Proteicas/metabolismoRESUMO
Protein kinase A anchoring proteins (AKAPs), defined by their capacity to target the cAMP-dependent protein kinase to distinct subcellular locations, function as molecular scaffolds mediating the assembly of multicomponent complexes to integrate and organise multiple signalling events. Despite their central importance in regulating cellular processes, little is known regarding their diverse structures and molecular mechanisms. Here, using bioinformatics and X-ray crystallography, we define a central domain of AKAP18 delta (AKAP18(CD)) as a member of the 2H phosphoesterase family. The domain features two conserved His-x-Thr motifs positioned at the base of a groove located between two lobes related by pseudo 2-fold symmetry. Nucleotide co-crystallisation screening revealed that this groove binds specifically to adenosine 5'-monophosphate (5'AMP) and cytosine 5'-monophosphate (5'CMP), with the affinity constant for AMP in the physiological concentration range. This is the first example of an AKAP capable of binding a small molecule. Our data generate two functional hypotheses for the AKAP18 central domain. It may act as a phosphoesterase, although we did not identify a substrate, or as an AMP sensor with the potential to couple intracellular AMP levels to PKA signalling events.