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

Liberación de sustancias en células senescentes

La invención trata de nanodispositivos para la liberación controlada de sustancias que comprenden un soporte recubierto por oligosacáridos, donde dichos oligosacáridos comprenden al menos 3 unidades de monosacáridos, y donde al menos uno de los monosacáridos es galactosa. Estos nanodispositivos liberan su carga de manera específica en células senescentes. La invención también recoge su procedimiento de obtención y sus usosPeer reviewedUniversidad Politécnica de Valencia, Consejo Superior de Investigaciones Científicas, Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina, Centro de Investigación Biomédica en Red de Enfermedades Rara

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

Dyskeratosis Congenita

Is a rare disease originally defined by a triad of mucocutaneous features: leukoplakia, reticulated skin pigmentation (observed at neck and upper chest), and nail dystrophy. Now is known to be a telomerase-defective disease with premature aging symptoms in different tissues (See “Aging Mechanisms” and “Aging Pathology”)

Structure of Dictyostelium discoideum telomeres. Analysis of possible replication mechanisms

© 2019 Rodriguez-Centeno et al.Telomeres are nucleo-protein structures that protect the ends of eukaryotic chromosomes. They are not completely synthesized during DNA replication and are elongated by specific mechanisms. The structure of the telomeres and the elongation mechanism have not been determined in Dictyostelium discoideum. This organism presents extrachromosomal palindromic elements containing two copies of the rDNA, also present at the end of the chromosomes. In this article the structure of the terminal region of the rDNA is shown to consist of repetitions of the A(G)n sequence where the number of Gs is variable. These repeats extend as a 3' single stranded region. The G-rich region is preceded by four tandem repetitions of two different DNA motifs. D. discoideum telomere reverse transcriptase homologous protein (TERTHP) presented RNase-sensitive enzymatic activity and was required to maintain telomere structure since terthp-mutant strains presented reorganizations of the DNA terminal regions. These modifications were different in several terthp-mutants and changed with their prolonged culture and subcloning. However, the terthp gene is not essential for D. discoideum proliferation. Telomeres could be maintained in terthp-mutant strains by homologous recombination mechanisms such as ALT (Alternative Lengthening of Telomeres) or HAATI (heterochromatin amplification-mediated and telomerase-independent). In agreement with this hypothesis, the expression of mRNAs coding for several proteins involved in homologous recombination was induced in terthp-mutant strains. Extrachromosomal rDNA could serve as substrate in these DNA homologous recombination reactions.RP and LS were funded by grants PI14-01495 and PI17-01401 (Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Spain supported by FEDER funds)

Telomerase deficiency and cancer susceptibility syndromes

Telomeres from most eukaryotes are composed of repeats of guanine-rich sequences whose main function is to preserve the end of the chromosomes. Telomeres are synthesised by a reverse transcriptase enzyme, telomerase (TERT), which forms part of a ribonucleoprotein complex containing also a RNA template molecule (TERC) and dyskerin. Exhaustion of telomeres during cell divisions triggers a DNA damage response that induces a senescence phenotype. The DNA damage machinery plays an essential role in maintaining the integrity of the genome and also detecting telomere shortening. However in some syndromes that involved mutations either in the telomerase complex genes or those involved in maintaining DNA secondary structure, such as the recQ helicase WRN, a higher frequency in the development of different types of malignancies is observed. We here describe the biology of some of these diseases, together with the molecular modifications in the telomerase complex genes and the impact of these alterations on the development of particular types of cancer. © 2009 Feseo.Peer Reviewe

Bionanopartículas biodegradables para liberación del péptido GSE24-2, procedimiento de obtención y utilización

[EN] The invention relates to biodegradable PLGA bionanoparticles encapsulating the GSE24-2 peptide with telomerase activity, and to pharmaceutical, cosmetic and biotechnological compositions comprising same. The pharmaceutical compositions are useful for treating diseases presenting a telomerase deficiency, such as dyskeratosis congenita, and diseases presenting ageing characteristics, such as Werner syndrome, Rothmund Thompson syndrome, and other diseases where there is DNA damage such as ataxia telangiectasia. The relation also relates to the method for producing said bionanoparticles by means of a w/o/w double emulsion technique.[FR] L'objet de l'invention concerne des bionanoparticules biodégradables de PLGA, dans lesquelles est encapsulé le peptide GSE24-2 à activité télomérase, et des compositions pharmaceutiques, cosmétiques, et biotechnologiques qui les contiennent. Les compositions pharmaceutiques sont utiles pour le traitement de maladies qui présentent un déficit en télomérase, comme la dyskératose congénitale, ainsi que des maladies qui se traduisent par un vieillissement, tel que le syndrome de Werner, de Rothmund Thompson, et d'autres maladies présentant des dommages de l'ADN comme l'ataxie télangiectasie. L'invention concerne également un procédé d'obtention desdites bionanoparticules au moyen d'une technique d'émulsion double w/o/w.[ES] El objeto de la invención se refiere a bionanopartículas biodegradables de PLGA en las cuales se ha encapsuladoel péptido GSE24-2con actividad telomerasa y composiciones farmacéuticas, cosméticas y biotecnológicas que las comprendan. Las composiciones farmacéuticas son útiles para el tratamiento de enfermedades que cursan con déficit telomerasa, como la disqueratosis congénita, así como enfermedades que cursen con envejecimiento, como el síndrome de Werner, de Rothmund Thompson, y de otras enfermedades donde haya un daño en el ADN como la ataxia telangiectasia. Igualmente, se describe el procedimiento de obtención de dichas bionanopartículas mediante una técnica de doble emulsión w/o/w.Peer reviewedConsejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Universidad del País Vasco, Centro de Investigaciones Biomédicas en Red de Enfermedades RarasA1 Solicitud de patente con informe sobre el estado de la técnic

Development and validation of a rapid HPLC method for the quantification of GSE4 peptide in biodegradable PEI-PLGA nanoparticles

et al.In this work a high performance liquid chromatographic (HPLC) method has been developed and validated for the content determination of GSE4 peptide in PEI-PLGA nanoparticles. Chromatographic separation was performed on a C18 column, and a gradient elution with a mobile phase composed of methanol and 0.1% aqueous trifluoroacetic acid (TFA) solution, at a flow rate of 1ml/min, was used. GSE4 peptide identification was made by fluorescence detection at 290nm. The elution of methanol:TFA was initially maintained at (20:80, v/v) for one min and the gradient changed to (80:20, v/v) in 6min. This ratio was then followed by isocratic elution at (80:20, v/v) during another min and for further 3min it was linearly modified to (20:80, v/v). The developed method was validated according to the ICH guidelines, being specific, linear in the range 10-100μg/ml (R2=0.9996), precise, exhibiting good inter-day and intra-day precision reflected by the relative standard deviation values (less than 3.88%), accurate, with a recovery rate of 100.18±0.95%, and stable for 48h at 5°C or at RT when encapsulated in nanoparticles. The method was simple, fast, and successfully used to determine the peptide content in GSE4-loaded PEI-PLGA nanoparticles.S.P. Egusquiaguirre thanks the Basque Government (Departamento de Educación, Universidades e Investigación) for the fellowship research grant. We also acknowledge financial support from the Spanish Government (Ministerio de Economia y Competitividad) for the Subprogram INNPACTO (IPT-2012-0674-090000) and FEDER funds.Peer Reviewe

FK506 sensitizes mammalian cells to high osmolarity by modulating p38 MAP kinase activation

The immunosuppressants tacrolimus (FK506) and cyclosporin A (CsA) have increased the survival rates in organ transplantation. Both drugs inhibit the protein phosphatase calcineurin (CaN) in activated T cells, exhibiting similar side-effects. Diabetes is observed more often in FK506 than CsA therapy, probably due to inhibition of new molecular targets other than CaN. We studied FK506 toxicity in mammalian cells. FK506, but not CsA, regulated p38 activation by osmotic stress, and decreased viability in osmostressed cells. In addition, FK506 treatment strongly increased the phosphorylation of the eukaryotic initiation factor-2α (eIF-2α) subunit. eIF-2α phosphorylation, p38 inhibition and cell lethality were relieved by addition of excess amino acids to the medium, suggesting that amino acid availability mediated FK506 toxicity. Therefore, these FK506-dependent responses could be relevant to the non-therapeutic effects of FK506 therapy.This study was supported by grants from Fondo de Investigaciones Sanitarias 00/0862, 01/1094 and 01/1626 and from FEDER 1FD87-1781.Peer Reviewe

p53 pathway activation by telomere attrition in X-DC primary fibroblasts occurs in the absence of ribosome biogenesis failure and as a consequence of DNA damage

[Background]: Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome with high clinical heterogeneity. Various mutations have been reported in DC patients, affecting genes that code for components of H/ACA ribonucleoproteins, proteins of the telomerase complex and components of the shelterin complex. [Objectives]: We aim to clarify the role of ribosome biogenesis failure in senescence induction in X-DC since some studies in animal models have reported a decrease in ribosome biogenesis as a major role in the disease. [Methods]: Dyskerin was depleted in normal human fibroblasts by expressing two DKC1 shRNAs. Common changes in gene expression profile between these dyskerin-depleted cells and X-DC fibroblasts were analyzed. [Results]: Dyskerin depletion induced early activation of the p53 pathway probably secondary to ribosome biogenesis failure. However, the p53 pathway in the fibroblasts from X-DC patients was activated only after an equivalent number of passes to AD-DC fibroblasts, in which telomere attrition in each division rendered shorter telomeres than control fibroblasts. Indeed, no induction of DNA damage was observed in dyskerin-depleted fibroblasts in contrast to X-DC or AD-DC fibroblasts suggesting that DNA damage induced by telomere attrition is responsible for p53 activation in X-DC and AD-DC fibroblasts. Moreover, p53 depletion in senescent DC fibroblasts rescued their proliferative capacity and reverted the morphological changes produced after prolonged culture. [Conclusions]: Our data indicate that ribosome biogenesis do not seem to play an important role in dyskeratosis congenita, conversely increasing DNA damage and activation of p53 pathway triggered by telomere shortening is the main activator of cell senescence.This work was supported by grants: 11/00949 from FIS and Fundación Ramón Areces. C. Manguán-García and J. Carrillo were supported by CIBER de Enfermedades Raras. WPeer Reviewe