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

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.This work was supported by grants PI1401495 (supported by FEDER funds) and ER15PR07ACC114/757 (Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III. Spain), 201320E075 (Consejo Superior de Investigaciones Científicas) and IPT-2012-0674- 090000 (Ministerio de Economía y Competitividad. Spain). CM-G is supported by the CIBER de Enfermedades Raras.Peer Reviewe

Molecular Diagnosis and Precision Therapeutic Approaches for Telomere Biology Disorders

Telomeres are nucleo-protein structures located at the end of chromosomes that protect them from degradation. Telomeres length is maintained by the activity of the telomerase complex. These structures are protected by a specialized protein complex named shelterin. In the absence of telomerase activity and/or protection telomeres are shortened after each round of DNA replication. When a critical size is reached, telomeres are recognized as damaged DNA by the cell p53-dependent DNA-repair system. Persistent activation of this pathway finally results in cell apoptosis or senescence

GSE4‐loaded nanoparticles a potential therapy for lung fibrosis that enhances pneumocyte growth, reduces apoptosis and DNA damage

Idiopathic pulmonary fibrosis is a lethal lung fibrotic disease, associated with aging with a mean survival of 2-5 years and no curative treatment. The GSE4 peptide is able to rescue cells from senescence, DNA and oxidative damage, inflammation, and induces telomerase activity. Here, we investigated the protective effect of GSE4 expression in vitro in rat alveolar epithelial cells (AECs), and in vivo in a bleomycin model of lung fibrosis. Bleomycin-injured rat AECs, expressing GSE4 or treated with GSE4-PLGA/PEI nanoparticles showed an increase of telomerase activity, decreased DNA damage, and decreased expression of IL6 and cleaved-caspase 3. In addition, these cells showed an inhibition in expression of fibrotic markers induced by TGF-β such as collagen-I and III among others. Furthermore, treatment with GSE4-PLGA/PEI nanoparticles in a rat model of bleomycin-induced fibrosis, increased telomerase activity and decreased DNA damage in proSP-C cells. Both in preventive and therapeutic protocols GSE4-PLGA/PEI nanoparticles prevented and attenuated lung damage monitored by SPECT-CT and inhibited collagen deposition. Lungs of rats treated with bleomycin and GSE4-PLGA/PEI nanoparticles showed reduced expression of α-SMA and pro-inflammatory cytokines, increased number of pro-SPC-multicellular structures and increased DNA synthesis in proSP-C cells, indicating therapeutic efficacy of GSE4-nanoparticles in experimental lung fibrosis and a possible curative treatment for lung fibrotic patients

Genetic analyses of aplastic anemia and idiopathic pulmonary fibrosis patients with short telomeres, possible implication of DNA-repair genes

Background: Telomeres are nucleoprotein structures present at the terminal region of the chromosomes. Mutations in genes coding for proteins involved in telomere maintenance are causative of a number of disorders known as telomeropathies. The genetic origin of these diseases is heterogeneous and has not been determined for a significant proportion of patients. Methods: This article describes the genetic characterization of a cohort of patients. Telomere length was determined by Southern blot and quantitative PCR. Nucleotide variants were analyzed either by high-resolution melting analysis and Sanger sequencing of selected exons or by massive sequencing of a panel of genes. Results: Forty-seven patients with telomere length below the 10% of normal population, affected with three telomeropathies: dyskeratosis congenita (4), aplastic anemia (22) or pulmonary fibrosis (21) were analyzed. Eighteen of these patients presented known pathogenic or novel possibly pathogenic variants in the telomere-related genes TERT, TERC, RTEL1, CTC1 and ACD. In addition, the analyses of a panel of 188 genes related to haematological disorders indicated that a relevant proportion of the patients (up to 35%) presented rare variants in genes related to DNA repair or in genes coding for proteins involved in the resolution of complex DNA structures, that participate in telomere replication. Mutations in some of these genes are causative of several syndromes previously associated to telomere shortening

Allosteric interactions at the M3 muscarinic acetylcholine receptor

The extracellular loops of muscarinic acetylcholine receptors are predicted to play a role in the binding and regulation of allosteric modulators. Furthermore, the sequence of the five subtypes of muscarinic receptors shows a large degree of diversity in this region. M3 receptor mutants, K523E, D518K and N132G, in which the substituted residues were those corresponding to the M1 subtype, were studied. As the amino acids in positions 518 and 523 are charged, the uncharged mutants, K523Q and D518N, were also created in order to observe any possible effect of charge. One question examined is whether these mutations changed the binding of orthosteric and allosteric ligands, generating a M1 receptor phenotype.Radioligand binding experiments revealed that one mutant, K523E, had a profound potentiating effect on the binding of prototypical modulators like gallamine, strychnine, brucine and N-chloromethylbrucine, but had minimal effects on the binding of a number of orthosteric ligands, including [3H]N-methylscopolamine ([3H]NMS) and acetylcholine (ACh). The increase in affinity was found at both the unoccupied and [3H]NMS-occupied receptors, with up to 70 fold increases in affinity being observed. Switches from negative to positive cooperativity for some strychnine-related compounds were found.At K523E, the affinities of the strychnine-related ligands were also increased up to 160 fold at the receptor-ACh complex, with up to 35 fold positive cooperativity being observed. Positive cooperativity of this magnitude is the highest that has been reported for M3 receptors.The dramatic changes in cooperativities and affinities of allosteric ligands at K523E did not result in generation of the M1 phenotype. The K523Q data suggest that the large changes in K523E result from the introduction of the negatively charged glutamate residue and not the loss of the positively charged lysine. The effect of K523E seems to be solely on the binding of allosteric ligands and the transmission of the effects of their binding to the orthosteric site.For the ligands acting at the gallamine site, all the effects of the allosteric modulators on ACh binding have been reproduced in functional studies, indicating that the allosteric modulation, seen in binding, is transmitted to the cellular response. A novel and unexpected finding is that WIN62,577 is an allosteric agonist at M3 muscarinic receptors and at K523E and N132G. The study also revealed that nanomolar concentrations of ACh may be present in assays of muscarinic receptor function and may give misleading interpretations of data. These artefacts were removed by preincubation with acetylcholinesterase, a control not previously used in functional studies of muscarinic receptors.The sensitivity of the binding of both orthosteric and allosteric ligands to the composition of the binding assay buffer has also been investigated in detail. In a phosphate buffer of low ionic strength (PB) the affinity constants of all the compounds studied, both orthosteric and allosteric, were increased, relative to a Hepes buffer of higher ionic strength, except for WIN 62,577, an allosteric ligand which binds to a different allosteric site from the prototypical modulators, and SVT-40776 a new M3 selective antagonist, indicating their different modes of binding. Cooperativities have also been switched from negative to positive by changing buffer.The two factors affecting the allosteric binding parameters of M3 receptors, PB and the mutation K523E, mutually potentiate each others effects. We have been able to obtain up to 10,000 fold changes in the affinity at the unoccupied receptor and 6400 fold increases in affinity at the ACh occupied receptor.The possible location of K523, relative to other residues on the external loops of muscarinic receptors shown to be important for the binding of allosteric ligands, has been explored using different models based on the X-ray structures of rhodopsin and the â2 adrenergic receptor

Werner syndrome

The Werner syndrome is a segmental progeroid syndrome of adult onset characterized by the presence of multiple features resembling accelerated aging accompanied by rare tumors. Commonly, the first symptom is the lack of growth spurt during one’s teens. Later on, WS patients have an aged appearance and early onset of age-related disorders; Werner’s syndrome is a rare disease caused by biallelic mutations in the WRN gene

GSE4, a small dyskerin- and GSE24.2-related peptide, induces telomerase and c-myc expression and reduces DNA damage and oxidative stress in dyskerin mutant cells

Resumen del póster presentado a la VIII Reunión Científica Anual del Centro de Investigación Biomédica En Red de Enfermedades Raras, celebrada en San Lorenzo del Escorial (Madrid) los días 12 y 13 de marzo de 2015.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 patients. The structural bases of GSE24.2 activity have been studied. The mutation of basic residues, possibly involved in nucleic acid binding, impaired activation of the c-myc and TERT promoters. 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 nucleolar localization signal impaired GSE24.2 activity. The biological activity of short peptides derived from GSE24.2 was also tested and one of them, GSE4, that probed to be active, was further characterized. Expression of this eleven amino acids long peptide increased telomerase activity and resulted in activation of c-myc and TERT promoters, increase of c-myc, TERT and TERC expression and reduction of DNA damage and oxidative stress in dyskerin-mutated cells. The level of biological activity of GSE4 was similar to that obtained by GSE24.2 expression. 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 pseudourydyl synthase domain present in GSE4 did not impair its activity, except for the activation of gene expression in dyskerin-mutated cells. These results indicated that GSE4 could be of great therapeutic interest for treatment of dyskeratosis congenita patients.Peer reviewe

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

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

Aluminium bronze alloys corrosion resistance guide

Reprint; originally publ. Jul 1981Available from British Library Document Supply Centre-DSC:7053.220(80) / BLDSC - British Library Document Supply CentreSIGLEGBUnited Kingdo