Effect of different ligands on BzdR-, Q1- or Q2-mediated repression of <i>P_N</i>.

<p><i>In vitro</i> transcription reactions were run as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057518#pone-0057518-g002" target="_blank">Figure 2B</a>, in the absence of repressor proteins (lane 2) or presence of 40 nM purified His₆-Q1 (lanes 3 to 6), His₆-Q2 (lanes 7 to 10) or His₆-BzdR (lanes 11 to 14). The ligands shikimate (S) (lanes 4, 8, 12), benzoyl-CoA (BCoA) (lanes 5, 9, 11), and benzoate (Bz) (lanes 6, 10) were added at 1 mM; ATP was added at 4 mM (lane 13). Lanes -, no ligand added. Lane 1, control assay without RNAP.</p

Modular architecture of the BzdR derivatives.

<p>Diagram showing the modular architecture of the BzdR protein, its NBzdR, NBzdRL and CBzdR domains, and the Q1, Q2 and Q1ΔL chimeras. The N-terminal domain (NBzdR), C-terminal domain (CBzdR) and linker region of BzdR are indicated in orange, blue and green, respectively. The <i>E. coli</i> SKI enzyme is shown in violet, and the Asp168Ala substitution in the Q2 chimera is indicated by an asterisk in red.</p

Bacterial strains and plasmids used in this work.

<p>Ap^r, ampicillin resistant; Cm^r chloramphenicol resistant; Km^r, kanamycin resistant; Nal^r, nalidixic acid; Rf^r, rifampicin resistant; Sm^r, streptomycin resistant; Sp^r, spectinomycin resistant.</p

The N-terminal domain of BzdR is a functional DNA-binding domain.

<p><b>A.</b> DNase I footprinting experiments were performed using the <i>P_N</i> probe and the purified regulators His₆-BzdR (control), His₆-NBzdR, and His₆-NBzdRL. The figure shows the results of footprinting assays conducted in the absence of the regulators (lane C), or the presence of 50, 100, or 200 nM of His₆-NBzdR (lanes 1 to 3, respectively) or His₆-NBzdRL (lanes 4 to 6, respectively). Lanes 7 and 8 are footprinting assays containing 50 and 100 nM of purified His₆-BzdR. Lane AG shows the A+G Maxam and Gilbert sequencing reaction. Protected regions (I, II, and III) are indicated with brackets. The −10 box and the transcription initiation site (+1) of the <i>P_N</i> promoter are also shown. Phosphodiester bonds hypersensitive to DNase I cleavage are indicated by asterisks. <b>B. </b><i>In vivo</i> effect of the N-terminal domain of BzdR on the repression of the <i>P_N</i> promoter. <i>E. coli</i> AFMCPN cells (containing a <i>P_N::lacZ</i> fusion chromosome insertion of the) harboring plasmid pCK01BzdR (BzdR), pCK01NBzdR (NBzdR) or pCK01NBzdRL (NBzdRL) or the control plasmid pCK01 (-), were grown anaerobically in LB medium until the mid-exponential culture phase. β-galactosidase activity is expressed in Miller units. Results from three independent experiments (<i>n</i> = 3) and errors bars are shown. <b>C.</b> Effect of BzdR, NBzdR and NBzdRL on <i>in vitro</i> transcription from <i>P_N</i>. Multiple-round <i>in vitro</i> transcription reactions were performed using the pJCD-P_N plasmid template, which produces a 184-nt mRNA from <i>P_N</i> (arrow), 50 nM <i>E. coli</i> RNAP, and 20 nM Fnr* activator. Transcription reactions were carried out in the absence of repressor (lane -) or presence of 40 nM purified His₆-BzdR (lanes BzdR), His₆-NBzdR (lanes NBzdR) or His₆-NBzdRL (lanes NBzdRL) and in the absence (-) or presence (+) of 2 mM benzoyl-CoA.</p

<i>In vitro</i> and <i>in vivo</i> effects of the Q1, Q1ΔL and Q2 chimeras on the <i>P_N</i> promoter.

<p>(<b>A</b>) DNase I footprinting experiments performed out using the <i>P_N</i> probe and the purified regulators His₆-BzdR (control) and His₆-Q1. The figure shows the results of footprinting assays conducted in the absence of the regulators (lane C), or presence of 25, 50, 100, and 200 nM of His₆-BzdR (lanes 1 to 4, respectively) or His₆-Q1 (lanes 5 to 8, respectively). Lane AG shows the A+G Maxam and Gilbert sequencing reaction. Protected regions (I, II, and III) are indicated by brackets. The -10 box and the transcription initiation site (+1) of the <i>P_N</i> promoter are also shown. Phosphodiester bonds hypersensitive to DNase I cleavage are indicated by asterisks. (<b>B</b>) <i>In vitro</i> effect of the Q1 and Q1ΔL chimeras on the activity of <i>P_N</i>. Multiple-round <i>in vitro</i> transcription reactions were performed using the pJCD-P_N plasmid template, which produces a 184-nt mRNA from <i>P_N</i> (arrow), 50 nM <i>E. coli</i> RNA polymerase (RNAP), and 20 nM Fnr* activator <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057518#pone.0057518-DuranteRodrguez2" target="_blank">[12]</a>. Transcription reactions were conducted in the absence of chimeric regulator (lane 1) or presence of 25 nM (lane 2) or 50 nM (lanes 3–12) of purified His₆-Q1 or His₆-Q1ΔL proteins. Shikimate (S) or shikimate plus ATP (S+ATP) were added at 1 mM (lanes 4 and 7), 2 mM (lanes 5 and 8) or 4 mM (lanes 6, 9, 11 and 12). (<b>C</b>) <i>In vivo</i> effect of BzdR (control) and the Q1 and Q2 chimeras on the activity of the <i>P_N</i> promoter. β-galactosidase activity (in Miller units) of <i>E. coli</i> MC4100 cells harboring plasmid pSJ3P_N (<i>P_N::lacZ</i>) and the plasmids pCK01BzdR (BzdR), pCK01Q1 (Q1), pCK01Q2 (Q2), or the control plasmid pCK01 (-). Cells were grown anaerobically until mid-exponential phase in LB medium supplemented, when indicated, with 5 mM shikimate (S). Results from three independent experiments (<i>n</i> = 3) and errors bars are shown. (<b>D</b>). <i>In vitro</i> effect of the Q2 chimera on the activity of <i>P_N</i>. <i>In vitro</i> transcription reactions were performed as in <i>panel B</i>, in the absence of Q2 (lane 1) or presence of 25 nM (lane 2) or 50 nM (lanes 3–9) of purified His₆-Q2 protein. Shikimate (S) or shikimate plus ATP (S+ATP) were added at 1 mM (lanes 4 and 7), 2 mM (lanes 5 and 8) or 4 mM (lanes 6 and 9).</p

GSE4, a Small Dyskerin- and GSE24.2-Related Peptide, Induces Telomerase Activity, Cell Proliferation and Reduces DNA Damage, Oxidative Stress and Cell Senescence in Dyskerin Mutant Cells

<div><p>Dyskeratosis congenita is an inherited disease caused by mutations in genes coding for telomeric components. It was previously reported that expression of a dyskerin-derived peptide, GSE24.2, increases telomerase activity, regulates gene expression and decreases DNA damage and oxidative stress in dyskeratosis congenita patient cells. The biological activity of short peptides derived from GSE24.2 was tested and one of them, GSE4, that probed to be active, was further characterized in this article. Expression of this eleven amino acids long peptide increased telomerase activity and reduced DNA damage, oxidative stress and cell senescence in dyskerin-mutated cells. GSE4 expression also activated c-myc and TERT promoters and increase of c-myc, TERT and TERC expression. The level of biological activity of GSE4 was similar to that obtained by GSE24.2 expression. Incorporation of a dyskerin nuclear localization signal to GSE24.2 did not change its activity on promoter regulation and DNA damage protection. However, incorporation of a signal that increases the rate of nucleolar localization impaired GSE24.2 activity. Incorporation of the dyskerin nuclear localization signal to GSE4 did not alter its biological activity. Mutation of the Aspartic Acid residue that is conserved in the pseudouridine synthase domain present in GSE4 did not impair its activity, except for the repression of c-myc promoter activity and the decrease of c-myc, TERT and TERC gene expression in dyskerin-mutated cells. These results indicated that GSE4 could be of great therapeutic interest for treatment of dyskeratosis congenita patients.</p></div

DNA-damage protective effect, telomerase activation and cell proliferation induction of one small peptide, GSE4, derived from GSE24.2.

<p>Panel A. One small peptide derived from GSE24.2, GSE4, and GSE24.2 were expressed in F9_A353V cells that were transfected with the pRRL-CMV-IRES-EGFP vector, either empty (Vector) or expressing GSE24.2 or GSE4. Twenty four hours later cells were lysed and the presence of γH2AX and α-tubulin (loading control) analyzed by western blot. Un-transfected F9 and F9-A353V cells were used as controls. The values at the bottom of the panel indicate the estimated ratio between γH2AX and α-tubulin expression levels referred to those found in cells transfected with the empty vector (F9-A353V vector). The amino acid sequences of GSE24.2 and GSE4 are indicated at the lower part of the panel. Panel B. The telomerase activity of F26IIB cells transfected with the pRRL-CMV-IRES-GFP vector empty (vector), expressing GSE24.2 (GSE24.2) or GSE4 (GSE4) was determined using the Telomeric Repeat Amplification Protocol (TRAP) assay. The amplification products obtained using three decreasing amounts of cell extracts for each cell line are shown in the right panel. Quantification of the amplification products, normalized to the internal control provided in the assay (indicated by an arrow at the right panel) is shown in the left panel. Panel C. Expression of Ki67 was determined by immunocytochemistry in F26IIB cells transfected as described in panel B. The percentage of cells expressing Ki67 is represented for each type of transfected cells. The experiments were repeated three times with similar results. Asterisks indicated the statistical significance (* p<0.05, **p<0.01, ***p<0.001).</p