Ataxia telangiectasia (AT) is a genetic disease caused by mutations in the ATM gene but the mechanisms underlying AT are not completely understood. Key functions of the ATM protein are to sense and ...regulate cellular redox status and to transduce DNA double-strand break signals to downstream effectors. ATM-deficient cells show increased ROS accumulation, activation of p38 protein kinase, and increased levels of DNA damage. GSE24.2 peptide and a short derivative GSE4 peptide corresponding to an internal domain of Dyskerin have proved to induce telomerase activity, decrease oxidative stress, and protect from DNA damage in dyskeratosis congenita (DC) cells. We have found that expression of GSE24.2 and GSE4 in human AT fibroblast is able to decrease DNA damage, detected by γ-H2A.X and 53BP1 foci. However, GSE24.2/GSE4 expression does not improve double-strand break signaling and repair caused by the lack of ATM activity. In contrast, they cause a decrease in 8-oxoguanine and OGG1-derived lesions, particularly at telomeres and mitochondrial DNA, as well as in reactive oxygen species, in parallel with increased expression of SOD1. These cells also showed lower levels of IL6 and decreased p38 phosphorylation, decreased senescence and increased ability to divide for longer times. Additionally, these cells are more resistant to treatment with H
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and the radiomimetic-drug bleomycin. Finally, we found shorter telomere length (TL) in AT cells, lower levels of TERT expression, and telomerase activity that were also partially reverted by GSE4. These observations suggest that GSE4 may be considered as a new therapy for the treatment of AT that counteracts the cellular effects of high ROS levels generated in AT cells and in addition increases telomerase activity contributing to increased cell proliferation.
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.
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.
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.
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.
Novel variants in telomere, DNA repair and replication genes are described that might indicate the contribution of variants in these genes to the development of telomeropathies. Patients carrying variants in telomere-related genes presented worse evolution after diagnosis than the rest of patients analyzed.
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.
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•Cationic and CPP-conjugated PLGA NPs encapsulating GSE24.2 peptide were developed.•PEI-PLGA, CPP5-PLGA and CPP6-PLGA-PEG NPs rescued the DNA damage in F9A353V cells.•These three ...formulations were able to recover the telomerase activity in VA13 cells.•These formulations are a promising approach to treat defective telomerase disorders.
The aim of the present study was to develop a novel strategy to deliver intracellularly the peptide GSE24.2 for the treatment of Dyskeratosis congenita (DC) and other defective telomerase disorders. For this purpose, biodegradable polymeric nanoparticles using poly(lactic-co-glycolic acid) (PLGA NPs) or poly(lactic-co-glycolic acid)-poly ethylene glycol (PLGA-PEG NPs) attached to either polycations or cell-penetrating peptides (CPPs) were prepared in order to increase their cellular uptake. The particles exhibited an adequate size and zeta potential, with good peptide loading and a biphasic pattern obtained in the in vitro release assay, showing an initial burst release and a later sustained release. GSE24.2 structural integrity after encapsulation was assessed using SDS–PAGE, revealing an unaltered peptide after the NPs elaboration. According to the cytotoxicity results, cell viability was not affected by uncoated polymeric NPs, but the incorporation of surface modifiers slightly decreased the viability of cells. The intracellular uptake exhibited a remarkable improvement of the internalization, when the NPs were conjugated to the CPPs. Finally, the bioactivity, addressed by measuring DNA damage rescue and telomerase reactivation, showed that some formulations had the lowest cytotoxicity and highest biological activity. These results proved that GSE24.2-loaded NPs could be delivered to cells, and therefore, become an effective approach for the treatment of DC and other defective telomerase syndromes.
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.
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.
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 3HN-methylscopolamine (3HNMS) and acetylcholine (ACh). The increase in affinity was found at both the unoccupied and 3HNMS-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.
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