RESUMEN
Early chronic lymphocytic leukaemia (CLL) is an ideal disease for immunotherapy. We previously showed that SEMG 1 is a cancer-testis (CT) antigen in CLL. In this study, SEMG 1 was applied as the bait in a yeast two-hybrid system of a testicular cDNA library. Seven clones were isolated and Protamine (Prm) 1 was identified as a novel CT antigen in early CLL. PRM1 transcripts were detected in 11/41 (26.8%) patients. Prm 1 protein was also expressed but heterogeneously within individual patients. Of the 11 patients expressing Prm 1, four expressed Zap 70 protein and seven did not. These results, therefore, indicate that Prm 1 could potentially be a suitable target for the design of tumour vaccine for patients with early CLL, including for those with poor risk CLL. High titres of Prm 1 IgG antibodies could be detected in 20 of these 41 CLL patients but not in any of the 20 healthy donors (P = 0.0001), suggesting the presence of Prm 1-reactive immune responses within the immune repertoire of patients with early CLL. Further work is warranted, especially in approaches to upregulate Prm 1 expression, and to determine the role of Prm 1 as an immunotherapeutic target for early CLL.
Asunto(s)
Biomarcadores de Tumor/análisis , Leucemia Linfocítica Crónica de Células B/inmunología , Protaminas/análisis , Anticuerpos/sangre , Linfocitos B/inmunología , Biomarcadores de Tumor/inmunología , Vacunas contra el Cáncer , Estudios de Casos y Controles , Detección Precoz del Cáncer , Ensayo de Inmunoadsorción Enzimática/métodos , Biblioteca de Genes , Humanos , Inmunoglobulina G/sangre , Inmunohistoquímica , Inmunoterapia , Leucemia Linfocítica Crónica de Células B/sangre , Masculino , Protaminas/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Proteínas de Secreción de la Vesícula Seminal/análisis , Testículo/inmunología , Técnicas del Sistema de Dos Híbridos , Proteína Tirosina Quinasa ZAP-70/análisisRESUMEN
Activated Akt has been previously implicated in acting on RS domain-containing proteins. However, it has been questioned whether its action is direct or it is mediated by co-existing SR kinase activity. To address this issue we studied in detail the phosphorylation of Lamin B Receptor (LBR) by Akt. Using synthetic peptides and a set of recombinant proteins expressing mutants of the LBR RS domain we now demonstrate that while all serines of the RS domain represent more or less equal phosphoacceptor sites for SRPK1, Ser80 and Ser82 are mainly targeted by Akt. 3D-modeling combined with molecular dynamics (MD) simulations show that amongst short, overlapping LBR RS-containing peptides complying with the minimum Akt recognition consensus sequence, only those bearing phosphosites either at Ser80 or Ser82 are able to fit into the active site of Akt, at least as effectively as its known substrate, GSK3-ß. Combined our results provide evidence that Akt kinases directly phosphorylate an RS domain-containing protein and that both the residues N-terminal the phosphosite and at position +1 are essential for Akt specificity, with the latter substrate position being compatible with the arginine residue of RS-repeats.
Asunto(s)
Arginina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Serina/metabolismo , Secuencia de Aminoácidos , Animales , Arginina/química , Arginina/genética , Sitios de Unión/genética , Glucógeno Sintasa Quinasa 3 beta/química , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Células HEK293 , Humanos , Immunoblotting , Simulación de Dinámica Molecular , Mutación , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Fosforilación , Unión Proteica , Dominios Proteicos , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/química , Proteínas Proto-Oncogénicas c-akt/genética , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/genética , Homología de Secuencia de Aminoácido , Serina/química , Serina/genética , Receptor de Lamina BRESUMEN
A significant amount of nuclear p53 is found associated with the nuclear matrix in cells that were exposed to genotoxic stress. In this study we identified Scaffold attachment factor B1 (SAFB1), a nuclear matrix-associated protein that binds the scaffold or matrix attachment regions (S/MARs) of genomic DNA, as a novel p53-interacting protein. SAFB1 was able to associate with p53 through its C-terminal domain, while significant co-localization of the two proteins was observed in cells treated with 5-fluorouracil or mithramycin. Binding of p53 to SAFB1 had a significant functional outcome, since SAFB1 was shown to suppress p53-mediated reporter gene expression. These data suggest that nuclear matrix-associated proteins may play a critical role in regulating p53 localization and activity.