CNBP: a multifunctional nucleic acid chaperone involved in cell death and proliferation control.

Cellular nucleic acid binding protein (CNBP) has been implicated in vertebrate craniofacial development and in myotonic dystrophy type 2 (DM2) and sporadic inclusion body myositis (sIBM) human diseases. In these seemingly unrelated biological processes, CNBP appears to be involved in controlling cell death and proliferation rates. Low levels of CNBP may reduce rate of global protein synthesis, thereby reducing proliferation and increasing apoptosis. Conversely, CNBP might affect transcription of genes required for cell proliferation. Experimental evidences gathered so far make it difficult to ascertain or rule out any of these possibilities. Moreover, both possibilities may not be mutually exclusive. CNBP is a small and strikingly conserved single-stranded nucleic acid binding protein that is able to bind DNA as well as RNA. CNBP has a broad spectrum of targets, ranging from regulatory sites in gene promoters to translational regulatory elements in mRNA untranslated regions. Biochemical experiments have recently shed light on the possible mechanism of action for CNBP, which may act as a nucleic acid chaperone catalyzing the rearrangement of G-rich nucleic acid secondary structures likely relevant for transcriptional and/or translational gene regulation. This review focuses on the involvement of CNBP in vertebrate craniofacial development and human DM2 and sIBM diseases, as well as on the biochemical and structural features of CNBP and its cellular and molecular mechanism of action.

Cellular nucleic-acid-binding protein, a transcriptional enhancer of c-Myc, promotes the formation of parallel G-quadruplexes.

G-rich sequences that contain stretches of tandem guanines can form four-stranded, intramolecular stable DNA structures called G-quadruplexes (termed G4s). Regulation of the equilibrium between single-stranded and G4 DNA in promoter regions is essential for control of gene expression in the cell. G4s are highly stable structures; however, their folding kinetics are slow under physiological conditions. CNBP (cellular nucleic-acid-binding protein) is a nucleic acid chaperone that binds the G4-forming G-rich sequence located within the NHE (nuclease hypersensitivity element) III of the c-Myc proto-oncogene promoter. Several reports have demonstrated that CNBP enhances the transcription of c-Myc in vitro and in vivo; however, none of these reports have assessed the molecular mechanisms responsible for this control. In the present study, by means of Taq polymerase stop assays, electrophoretic mobility-shift assays and CD spectroscopy, we show that CNBP promotes the formation of parallel G4s to the detriment of anti-parallel G4s, and its nucleic acid chaperone activity is required for this effect. These findings are the first to implicate CNBP as a G4-folding modulator and, furthermore, assign CNBP a novel mode-of-action during c-Myc transcriptional regulation.

Dissecting CNBP, a zinc-finger protein required for neural crest development, in its structural and functional domains.

Cellular nucleic-acid-binding protein (CNBP) plays an essential role in forebrain and craniofacial development by controlling cell proliferation and survival to mediate neural crest expansion. CNBP binds to single-stranded nucleic acids and displays nucleic acid chaperone activity in vitro. The CNBP family shows a conserved modular organization of seven Zn knuckles and an arginine-glycine-glycine (RGG) box between the first and second Zn knuckles. The participation of these structural motifs in CNBP biochemical activities has still not been addressed. Here, we describe the generation of CNBP mutants that dissect the protein into regions with structurally and functionally distinct properties. Mutagenesis approaches were followed to generate: (i) an amino acid replacement that disrupted the fifth Zn knuckle; (ii) N-terminal deletions that removed the first Zn knuckle and the RGG box, or the RGG box alone; and (iii) a C-terminal deletion that eliminated the three last Zn knuckles. Mutant proteins were overexpressed in Escherichia coli, purified, and used to analyze their biochemical features in vitro, or overexpressed in Xenopus laevis embryos to study their function in vivo during neural crest cell development. We found that the Zn knuckles are required, but not individually essential, for CNBP biochemical activities, whereas the RGG box is essential for RNA-protein binding and nucleic acid chaperone activity. Removal of the RGG box allowed CNBP to preserve a weak single-stranded-DNA-binding capability. A mutant mimicking the natural N-terminal proteolytic CNBP form behaved as the RGG-deleted mutant. By gain-of-function and loss-of-function experiments in Xenopus embryos, we confirmed the participation of CNBP in neural crest development, and we demonstrated that the CNBP mutants lacking the N-terminal region or the RGG box alone may act as dominant negatives in vivo. Based on these data, we speculate about the existence of a specific proteolytic mechanism for the regulation of CNBP biochemical activities during neural crest development.

Signaling modulation of bile salt-induced necrosis in isolated rat hepatocytes.

Hydrophobic bile salts induce either necrosis or apoptosis depending on the severity of the injury caused by them. Since bile salt-induced apoptosis is influenced by Ca2+- and protein kinase-signaling pathways, and both necrosis and apoptosis share common initiating mechanisms, we analyzed whether these signaling cascades also influence bile salt-induced necrosis in isolated rat hepatocytes. Taurochenodeoxycholate (TCDC, 0.25-1.50 mM, 2 h) reduced, in a dose-dependent manner, the percentage of viable hepatocytes, and increased the release of the cytosolic enzyme, lactate dehydrogenase (LDH) and alanine aminotransferase (ALAT), and that of the plasma membrane enzyme, alkaline phosphatase (AP). The PKC inhibitors, H7 (100 microM) and chelerythrine (2.5 microM), both prevented significantly TCDC-induced necrosis. On the contrary, the PKA activator, dibutyryl-cAMP, exacerbated TCDC-induced cell damage in a dose-dependent manner; this effect was more likely due to cAMP-mediated PKA activation, as the PKA inhibitor, KT5720 (1 microM), counteracted this effect. Instead, the intracellular Ca2+ chelator, BAPTA/AM (20 microM), was without effect. TCDC (1 mM) increased lipid peroxidation from 0.7 +/- 0.2 to 7.5 +/- 0.9 nmol of malondialdehyde per mg of protein, p < 0.001; the addition of the free radical scavenger, diphenyl-p-phenylendiamine, completely blocked this increase and prevented significantly TCDC-induced necrosis. PKC inhibition induced only a slight attenuation of TCDC-induced lipid peroxidation. Possible mechanisms accounting for the modulatory effect of signal transduction pathways on TCDC-induced necrosis, including signaling influence on TCDC transport events and TCDC-induced oxidative stress, are discussed.

[Non conventional virological markers in HIV-infected patients: T-HIV DNA, 2LTR-HIV DNA and HIV RNA]. / Marcadores virológicos no convencionales en pacientes infectados con el virus de la inmunodeficiencia humana: ADN HIV-T, ADN HIV-2LTR y ARN de HIV.

Highly active antiretroviral therapy (HAART) induces a persistent reduction of the plasmatic viremia, contributing to decrease mortality and morbidity of infected people with human immunodeficiency virus (HIV). Thus, viral load (VL) is the reference method to evaluate therapy effectiveness. However, even in the presence of efficient HAART viral eradication was yet not achieved. In this study, we analyzed the presence of total HIV DNA (T-HIV DNA), non-integrated DNA with 2LTR (2LTR-HIV DNA) and HIV RNA in a group of 55 HIV-positive subjects from Rosario City, with different clinical stages, with and without HAART. All markers were evaluated by PCR assays optimized in our laboratory that included colorimetric detection in microplate. HIV RNA clinical sensitivity was compared with a reference test, bDNA, resulting in 74% and 64% respectively, with an 85% of agreement. Thus, our HIV RNA assay could be used to monitor patients under HAART and at risk of infection. The 2LTR-HIV DNA was 54% positive although it was absent in patients with high VL. This marker was considered a labile product therefore its presence was associated with recent infection. However, current evidences question its stability. Thus, its clinical significance should be reconsidered. The absence of 2LTR-HIV DNA in patients with detectable VL may relate to the heterogeneity of the sequence used for its detection. T-HIV DNA was present in 100% of the samples and could be a relevant remission marker when therapies that effectively eradicate the infection became available.

Marcadores virologicos no convencionales en pacientes infectados con el virus de la inmunodeficiencia humana: ADN HIV-T, ADN HIV-2LTR y ARN de HIV / Non conventional virological markers in HIV-infected patients: T-HIV DNA, 2LTR-HIV DNA and HIV RNA

La terapia antirretrovial de alta eficacia (TAAE) induce una reducción marcada y persisatente de la viremia plasmática, contribuvendo a disminuir la mortalidad de los pacientes HIV-positivos. Así, la carga viral (CV) es el método de referencia para evaluar la eficacia terapéutica. Sin embargo, aun en presencia de una TAAE eficiente no se ha logrado la erradicación viral. En este estudio analizamos la presencia del ADN total de HIV (ADN HIV-T), del ADN no integrado con 2LTR (ADN HIV-2LTR) y del ARN de HIV, en un grupo de 55 pacientes HIV-positivos en distintos estadios clínicos, con y sin TAAE, mediante ensayos de PCR con revelado colorimétrico en microplaca, optimizados en nuestro laboratorio. La sensibilidad clínica de ARN del HIV fue evaluada con el bDNA, resultando del 74% y del 64%, respectivamente, con una concordancia del 85%. Este ensayo podría utilizado en el seguimiento de pacientes bajo TAAE. EI ADN HIV-2LTR resultó positivo en el 54% aunque estuvo ausente en pacientes con elevada CV. Este marcador se considereba un producto lábil y su presencia se asociaba a infección reciente. Sin embargo, actuales evidencias ponen en discusión su estabilidad por lo que su significado clínico debe ser reconsiderado. La ausencia del ADN HIV-2LTR en pacientes con CV detectable puede relacionarse con la heterogeneidade de la secuencia utilizada para su detección. EI ADN HIV-T estuvo presente en el 100% de las muestras y resultaría relevante como marcador de remisión cuando se dispongan de terapias que efectivamente erradiquen la infección.

Marcadores virologicos no convencionales en pacientes infectados con el virus de la inmunodeficiencia humana: ADN HIV-T, ADN HIV-2LTR y ARN de HIV / Non conventional virological markers in HIV-infected patients: T-HIV DNA, 2LTR-HIV DNA and HIV RNA

La terapia antirretrovial de alta eficacia (TAAE) induce una reducción marcada y persisatente de la viremia plasmática, contribuvendo a disminuir la mortalidad de los pacientes HIV-positivos. Así, la carga viral (CV) es el método de referencia para evaluar la eficacia terapéutica. Sin embargo, aun en presencia de una TAAE eficiente no se ha logrado la erradicación viral. En este estudio analizamos la presencia del ADN total de HIV (ADN HIV-T), del ADN no integrado con 2LTR (ADN HIV-2LTR) y del ARN de HIV, en un grupo de 55 pacientes HIV-positivos en distintos estadios clínicos, con y sin TAAE, mediante ensayos de PCR con revelado colorimétrico en microplaca, optimizados en nuestro laboratorio. La sensibilidad clínica de ARN del HIV fue evaluada con el bDNA, resultando del 74% y del 64%, respectivamente, con una concordancia del 85%. Este ensayo podría utilizado en el seguimiento de pacientes bajo TAAE. EI ADN HIV-2LTR resultó positivo en el 54% aunque estuvo ausente en pacientes con elevada CV. Este marcador se considereba un producto lábil y su presencia se asociaba a infección reciente. Sin embargo, actuales evidencias ponen en discusión su estabilidad por lo que su significado clínico debe ser reconsiderado. La ausencia del ADN HIV-2LTR en pacientes con CV detectable puede relacionarse con la heterogeneidade de la secuencia utilizada para su detección. EI ADN HIV-T estuvo presente en el 100% de las muestras y resultaría relevante como marcador de remisión cuando se dispongan de terapias que efectivamente erradiquen la infección. (AU)

Marcadores virológicos no convencionales en pacientes infectados con el virus de la inmunodeficiencia humana: ADN HIV-T, ADN HIV-2LTR y ARN de HIV. / [Non conventional virological markers in HIV-infected patients: T-HIV DNA, 2LTR-HIV DNA and HIV RNA]

Highly active antiretroviral therapy (HAART) induces a persistent reduction of the plasmatic viremia, contributing to decrease mortality and morbidity of infected people with human immunodeficiency virus (HIV). Thus, viral load (VL) is the reference method to evaluate therapy effectiveness. However, even in the presence of efficient HAART viral eradication was yet not achieved. In this study, we analyzed the presence of total HIV DNA (T-HIV DNA), non-integrated DNA with 2LTR (2LTR-HIV DNA) and HIV RNA in a group of 55 HIV-positive subjects from Rosario City, with different clinical stages, with and without HAART. All markers were evaluated by PCR assays optimized in our laboratory that included colorimetric detection in microplate. HIV RNA clinical sensitivity was compared with a reference test, bDNA, resulting in 74

and 64

respectively, with an 85

of agreement. Thus, our HIV RNA assay could be used to monitor patients under HAART and at risk of infection. The 2LTR-HIV DNA was 54

positive although it was absent in patients with high VL. This marker was considered a labile product therefore its presence was associated with recent infection. However, current evidences question its stability. Thus, its clinical significance should be reconsidered. The absence of 2LTR-HIV DNA in patients with detectable VL may relate to the heterogeneity of the sequence used for its detection. T-HIV DNA was present in 100

of the samples and could be a relevant remission marker when therapies that effectively eradicate the infection became available.