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1.
Biochem Biophys Res Commun ; 406(3): 383-8, 2011 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-21329658

RESUMO

Cargos destined to enter or leave the cell nucleus are typically transported by receptors of the importin ß family to pass the nuclear pore complex. The yeast Saccharomyces cerevisiae comprises 14 members of this protein family, which can be divided in importins and exportins. The Ran GTPase regulates the association and dissociation of receptors and cargos as well as the transport direction through the nuclear pore. All receptors bind to Ran exclusively in its GTP-bound state and this event is restricted to the nuclear compartment. We determined the Ran-GTP binding properties of all yeast transport receptors by biosensor measurements and observed that the affinity of importins for Ran-GTP differs significantly. The dissociation constants range from 230 pM to 270 nM, which is mostly based on a variability of the off-rate constants. The divergent affinity of importins for Ran-GTP suggests the existence of a novel mode of nucleocytoplasmic transport regulation. Furthermore, the cellular concentration of ß-receptors and of other Ran-binding proteins was determined. We found that the number of ß-receptors altogether about equals the amounts of yeast Ran, but Ran-GTP is not limiting in the nucleus. The implications of our results for nucleocytoplasmic transport mechanisms are discussed.


Assuntos
Núcleo Celular/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , beta Carioferinas/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Transporte Ativo do Núcleo Celular , Guanosina Trifosfato/metabolismo
2.
AIDS Res Hum Retroviruses ; 24(5): 717-23, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18462078

RESUMO

A few years ago, reactivation of human endogenous retrovirus K (HERV-K) proviruses in melanoma was described. The expression of HERV-K proteins induces humoral immune responses. The aim of the present study was to elucidate the prognostic relevance of serological anti-HERV-K reactivity in melanoma patients. In a retrospective study, anti-HERV-K Gag and Env antibodies were detected in 51 of the 312 randomly selected and blinded sera from melanoma patients, but not in any of the 70 sera from healthy controls. Comparing serological HERV-K reactivity with established melanoma markers revealed a significant correlation (p = 0.018, Chi-square test) with the stage of disease classified according to the American Joint Committee on Cancer (AJCC). Anti-HERV-K reactivity was elevated in patients with acrolentiginous/mucosal/uveal melanoma (tumor subtypes developing at sun-protected sites) compared to patients with lentigo/nodular/superficial spreading melanoma (p = 0.011, Chi-square test). Patients with anti-HERV-K antibodies had a significantly decreased disease-specific overall survival (stage I-IV, p < 0.001; stage I-III, p = 0.005, log-rank test). Significantly, multivariate Cox regression analysis including prognostic markers in clinical use (e.g., AJCC stage, T-class, serum level of S100-beta) revealed serological HERV-K reactivity as an independent marker of reduced survival probability (p = 0.027) in melanoma patients with the early stages of the disease (AJCC I-III). This is the first report that the humoral anti-HERV-K immune response may provide additional prognostic information to that of established melanoma markers.


Assuntos
Anticorpos Antivirais/sangue , Retrovirus Endógenos/imunologia , Melanoma/sangue , Melanoma/diagnóstico , Neoplasias Cutâneas/sangue , Neoplasias Cutâneas/diagnóstico , Neoplasias Uveais/sangue , Neoplasias Uveais/diagnóstico , Adulto , Idoso , Biomarcadores/sangue , Feminino , Produtos do Gene gag/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Fatores de Crescimento Neural/sangue , Probabilidade , Prognóstico , Estudos Retrospectivos , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/sangue , Proteínas do Envelope Viral/imunologia
3.
J Mol Biol ; 379(4): 678-94, 2008 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-18485366

RESUMO

Proteins can enter the nucleus through various receptor-mediated import pathways. One class of import cargos carries a classical nuclear localization signal (cNLS) containing a short cluster of basic residues. This pathway involves importin alpha (Impalpha), which possesses the cNLS binding site, and importin beta (Impbeta), which translocates the import complex through the nuclear pore complex. The defining criteria for a cNLS protein from Saccharomyces cerevisiae are an in vivo import defect in Impalpha and Impbeta mutants, direct binding to purified Impalpha, and stimulation of this binding by Impbeta. We show for the first time that endogenous S. cerevisiae proteins Prp20, Cdc6, Swi5, Cdc45, and Clb2 fulfill all of these criteria identifying them as authentic yeast cNLS cargos. Furthermore, we found that the targeting signal of Prp20 is a bipartite cNLS and that of Cdc6 is a monopartite cNLS. Basic residues present within these motifs are of different significance for the interaction with Impalpha. We determined the binding constants for import complexes containing the five cNLS proteins by surface plasmon resonance spectrometry. The dissociation constants for cNLS/alpha/beta complexes differ considerably, ranging from 1 nM for Cdc6 to 112 nM for Swi5, suggesting that the nuclear import kinetics is determined by the strength of cNLS/Impalpha binding. Impbeta enhances the affinity of Impalpha for cNLSs approximately 100-fold. This stimulation of cNLS binding to Impalpha results from a faster association in the presence of Impbeta, whereas the dissociation rate is unaffected by Impbeta. This implies that, after entry into the nucleus, the release of Impbeta by the Ran guanosine triphosphatase (Ran GTPase) from the import complex is not sufficient to dissociate the cNLS/Impalpha subcomplex. Our observation that the nucleoporin Nup2, which had been previously shown to release the cNLS from Impalpha in vitro, is required for efficient import of all the genuine cNLS cargos supports a general role of Nup2 in import termination.


Assuntos
Sinais de Localização Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes Fúngicos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Carioferinas/genética , Carioferinas/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Sinais de Localização Nuclear/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , beta Carioferinas/genética , beta Carioferinas/metabolismo
4.
Nucleic Acids Res ; 36(2): 648-65, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18073200

RESUMO

LINE-1 (L1) is a highly successful autonomous non-LTR retrotransposon and a major force shaping mammalian genomes. Although there are about 600 000 L1 copies covering 23% of the rat genome, full-length rat L1s (L1Rn) with intact open reading frames (ORFs) representing functional master copies for retrotransposition have not been identified yet. In conjunction with studies to elucidate the role of L1 retrotransposons in tumorigenesis, we isolated and characterized 10 different cDNAs from transcribed full-length L1Rn elements in rat chloroleukemia (RCL) cells, each encoding intact ORF1 proteins (ORF1p). We identified the first functional L1Rn retrotransposon from this pool of cDNAs, determined its activity in HeLa cells and in the RCL cell line the cDNAs originated from and demonstrate that it is mobilized in the tumor cell line in which it is expressed. Furthermore, we generated monoclonal antibodies directed against L1Rn ORF1 and ORF2-encoded recombinant proteins, analyzed the expression of L1-encoded proteins and found ORF1p predominantly in the nucleus. Our results support the hypothesis that the reported explosive amplification of genomic L1Rn sequences after their transcriptional activation in RCL cells is based on L1 retrotransposition. Therefore, L1 activity might be one cause for genomic instability observed during the progression of leukemia.


Assuntos
Leucemia Experimental/genética , Elementos Nucleotídeos Longos e Dispersos , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Núcleo Celular/química , Citoplasma/química , DNA Complementar/isolamento & purificação , Células HeLa , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta , Poliadenilação , Proteínas/análise , Proteínas/genética , Proteínas/metabolismo , Ratos , Transcrição Gênica
5.
Melanoma Res ; 16(3): 223-34, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16718269

RESUMO

The human endogenous retrovirus-K encodes two potential tumor proteins, Rec and Np9. Rec is related to the Rev protein of HIV-1 and has been shown to be associated with tumor development in nude mice. Having shown the expression of human endogenous retrovirus-K in human melanomas and melanoma cell lines, tools were developed to allow the expression of the transmembrane envelope, Rec and Np9 mRNA and proteins to be studied in more detail. The expression of spliced env, rec and np9 was investigated by reverse transcriptase-polymerase chain reaction using a set of primers developed to discriminate between full-length and spliced mRNA. Env-specific, Rec-specific and Np9-specific antisera were produced, characterized and used to study protein expression in melanomas and melanoma cell lines by immunohistochemistry, immunofluorescence and Western blot analyses. Existence of human endogenous retrovirus-K Rec and Np9-specific antibodies in the sera of melanoma patients were analyzed by Western blot of immunofluorescence studies. The expression of both spliced env and rec mRNA was detected in 39% of the melanomas and in 40% of the melanoma cell lines and np9 mRNA was detected in 29 and 21%, respectively. In normal neonatal melanocytes, spliced rec mRNA was detected in the absence of spliced env mRNA. Using antisera specific for Rec and Np9, Rec protein was found in 14% of the melanomas but Np9 in none. In addition, cell surface expression of the putatively immunosuppressive transmembrane envelope protein and release of virus particles were shown. Antibodies specific for neither Rec nor Np9 were detected. The transmembrane envelope protein, Rec and Np9 proteins are expressed in melanoma cells with a pattern similar to that seen in teratocarcinoma cell lines. Additional experiments are needed to determine their involvement, if any, in cell proliferation and tumor progression.


Assuntos
Retrovirus Endógenos/isolamento & purificação , Produtos do Gene env/biossíntese , Melanoma/metabolismo , Melanoma/virologia , Proteínas do Envelope Viral/biossíntese , Sequência de Aminoácidos , Western Blotting , Retrovirus Endógenos/genética , Retrovirus Endógenos/crescimento & desenvolvimento , Retrovirus Endógenos/imunologia , Mapeamento de Epitopos/métodos , Imunofluorescência/métodos , Expressão Gênica , Produtos do Gene env/genética , Genes env , Células HeLa , Humanos , Soros Imunes/imunologia , Melanoma/genética , Melanoma/patologia , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia
6.
Cancer Res ; 66(3): 1658-63, 2006 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-16452225

RESUMO

We previously described the expression of melanoma-associated endogenous retrovirus (MERV) proteins and viral particles in human melanomas and metastases. The objective of the present study was to determine whether a humoral immune response to MERV proteins occurs in melanoma. Candidate B-cell epitopes on MERV proteins were predicted using bioinformatic screening. The reactivity of MERV peptides corresponding to the predicted epitopes with antibodies prevalent in sera of melanoma patients was analyzed. An immunodominant peptide located in the env protein of MERV was identified. Subsequent analyzes using 81 samples from stage I to stage IV melanoma patients and 95 sera from healthy subjects revealed statistically significant differences in seroprevalence of antibodies in melanoma sera samples when compared with reference samples from healthy subjects. The prevalence of anti-MERV antibodies in melanoma patient sera was confirmed by immunofluorescence on env-transfected cells. These data indicate the potential of this candidate peptide as target for diagnosis and immunotherapy.


Assuntos
Biomarcadores Tumorais/imunologia , Retrovirus Endógenos/imunologia , Melanoma/virologia , Proteínas do Envelope Viral/imunologia , Anticorpos Antineoplásicos/sangue , Anticorpos Antineoplásicos/imunologia , Reações Cruzadas , Epitopos de Linfócito B/imunologia , Células HeLa , Humanos , Epitopos Imunodominantes/imunologia , Melanoma/sangue , Melanoma/imunologia , Melanoma/patologia , Estadiamento de Neoplasias
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