Protein Isoaspartate Methyltransferase Prevents Apoptosis Induced by Oxidative Stress in Endothelial Cells: Role of Bcl-Xl Deamidation and Methylation

BACKGROUND:Natural proteins undergo in vivo spontaneous post-biosynthetic deamidation of specific asparagine residues with isoaspartyl formation. Deamidated-isomerized molecules are both structurally and functionally altered. The enzyme isoaspartyl protein carboxyl-O-methyltransferase (PCMT; EC 2.1.1.77) has peculiar substrate specificity towards these deamidated proteins. It catalyzes methyl esterification of the free alpha-carboxyl group at the isoaspartyl site, thus initiating the repair of these abnormal proteins through the conversion of the isopeptide bond into a normal alpha-peptide bond. Deamidation occurs slowly during cellular and molecular aging, being accelerated by physical-chemical stresses brought to the living cells. Previous evidence supports a role of protein deamidation in the acquisition of susceptibility to apoptosis. Aim of this work was to shed a light on the role of PCMT in apoptosis clarifying the relevant mechanism(s). METHODOLOGY/PRINCIPAL FINDINGS:Endothelial cells transiently transfected with various constructs of PCMT, i.e. overexpressing wild type PCMT or negative dominants, were used to investigate the role of protein methylation during apoptosis induced by oxidative stress (H(2)O(2); 0.1-0.5 mM range). Results show that A) Cells overexpressing "wild type" human PCMT were resistant to apoptosis, whereas overexpression of antisense PCMT induces high sensitivity to apoptosis even at low H(2)O(2) concentrations. B) PCMT protective effect is specifically due to its methyltransferase activity rather than to any other non-enzymatic interactions. In fact negative dominants, overexpressing PCMT mutants devoid of catalytic activity do not prevent apoptosis. C) Cells transfected with antisense PCMT, or overexpressing a PCMT mutant, accumulate isoaspartyl-containing damaged proteins upon H(2)O(2) treatment. Proteomics allowed the identification of proteins, which are both PCMT substrates and apoptosis effectors, whose deamidation occurs under oxidative stress conditions leading to programmed cell death. These proteins, including Hsp70, Hsp90, actin, and Bcl-xL, are recognized and methylated by PCMT, according to the general repair mechanism of this methyltransferase. CONCLUSION/SIGNIFICANCE:Apoptosis can be modulated by "on/off" switch partitioning the amount of specific protein effectors, which are either in their active (native) or inactive (deamidated) molecular forms. Deamidated proteins can also be functionally restored through methylation. Bcl-xL provides a case for the role of PCMT in the maintenance of functional stability of this antiapoptotic protein

SIRT1 contributes to telomere maintenance and augments global homologous recombination

SIRT1 is a positive regulator of telomere length and attenuates age-associated telomere shortening

SIRT1 Is Necessary for Proficient Telomere Elongation and Genomic Stability of Induced Pluripotent Stem Cells

The NAD-dependent deacetylase SIRT1 is involved in chromatin silencing and genome stability. Elevated SIRT1 levels in embryonic stem cells also suggest a role for SIRT1 in pluripotency. Murine SIRT1 attenuates telomere attrition in vivo and is recruited at telomeres in induced pluripotent stem cells (iPSCs). Because telomere elongation is an iPSC hallmark, we set out to study the role of SIRT1 in pluripotency in the setting of murine embryonic fibroblasts reprogramming into iPSCs. We find that SIRT1 is required for efficient postreprogramming telomere elongation, and that this effect is mediated by a c-MYC-dependent regulation of the mTert gene. We further demonstrate that SIRT1-deficient iPSCs accumulate chromosomal aberrations and show a derepression of telomeric heterochromatin. Finally, SIRT1-deficient iPSCs form larger teratomas that are poorly differentiated, highlighting a role for SIRT1 in exit from pluripotency. In summary, this work demonstrates a role for SIRT1 in the maintenance of pluripotency and modulation of differentiation

Identification of TERRA locus unveils a telomere protection role through association to nearly all chromosomes.

Telomeric RNAs (TERRAs) are UUAGGG repeat-containing RNAs that are transcribed from the subtelomere towards the telomere. The precise genomic origin of TERRA has remained elusive. Using a whole-genome RNA-sequencing approach, we identify novel mouse transcripts arising mainly from the subtelomere of chromosome 18, and to a lesser extend chromosome 9, that resemble TERRA in several key aspects. Those transcripts contain UUAGGG-repeats and are heterogeneous in size, fluctuate in abundance in a TERRA-like manner during the cell cycle, are bound by TERRA RNA-binding proteins and are regulated in a manner similar to TERRA in response to stress and the induction of pluripotency. These transcripts are also found to associate with nearly all chromosome ends and downregulation of the transcripts that originate from chromosome 18 causes a reduction in TERRA abundance. Interestingly, downregulation of either chromosome 18 transcripts or TERRA results in increased number of telomere dysfunction-induced foci, suggesting a protective role at telomeres.We are indebted to Stefan Schoeftner and Susana Llanos for reagents and to Manuel Serrano, Maria Elisa Varela and Antonio Maraver for very helpful suggestions and discussion on the manuscript. We thank Diego Megias for confocal image acquisition and to Miguel Angel Grillo, Maria del Carmen Carralero and Juan Cruz Cigudosa for the Spectral Karyotyping (SKY). We thank Luis E. Donate for manuscript preparation. External RNAseq data were generated and analysed by the UW ENCODE group and by the transcriptome group at Cold Spring Harbor Laboratories and the Center for Genomic Regulation (CRG in Barcelona), who are participants in the ENCODE Transcriptome Group. ChIP data of transcription factors binding site were generated and analysed by the laboratories of Michael Snyder at Stanford University and Sherman Weissman at Yale University within the ENCODE Project. Histone marks data belong to the Caltech/ENCODE project in which cell growth, ChIP and Illumina library construction were done in the laboratory of Barbara Wold (California Institute of Technology). Sequencing was done at the Millard and Muriel Jacobs Genetics and Genomics Laboratory at the California Institute of Technology, initial HiSeq data were generated at Illumina Inc., Hawyard, CA. Cell growth and ChIP of histone marks were carried out by Georgi Marinov, Katherine Fisher, Gordon Kwan, Antony Kirilusha, Ali Mortazavi, Gilberto DeSalvo and Brian Williams. Library Construction, Sequencing and Primary Data Handling by Lorianne Schaeffer, Diane Trout, Igor Antoschechkin (California Institute of Technology), Lu Zhang and Gary Schroth (Illumina Inc.). Data processing and submission by Georgi Marinov and Diane Trout. Research in the Blasco laboratory is funded by the Spanish Ministry of Economy and Competitiveness Projects SAF2008-05384 and CSD2007-00017, the Madrid Regional Government Project S2010/BMD-2303 (ReCaRe), the European Union FP7 Project FHEALTH-2010-259749 (EuroBATS), the European Research Council (ERC) Project GA#232854 (TEL STEM CELL), the Preclinical Research Award from Fundacion Lilly (Spain), Fundacion Botin (Spain) and AXA Research Fund.S

Time-to-event interpretable machine learning for multiple sclerosis worsening prediction: results from iDPP@ CLEF 2023

In this work, we present a framework for the interpretable analysis of machine learning algorithms to predict the Multiple Sclerosis worsening using the datasets provided by the iDPP@ CLEF 2023 Challenge. The proposed framework is modular and allows to investigate the link between the provided static and dynamic features and the outcome to be predicted. Our findings show that better performance could be achieved by using Random Survival Forests together with temporal information about the clinical scores and a proposed feature related to the normalized frequency of patients’ relapses

MeCP2 Dependent Heterochromatin Reorganization during Neural Differentiation of a Novel Mecp2-Deficient Embryonic Stem Cell Reporter Line.

The X-linked Mecp2 is a known interpreter of epigenetic information and mutated in Rett syndrome, a complex neurological disease. MeCP2 recruits HDAC complexes to chromatin thereby modulating gene expression and, importantly regulates higher order heterochromatin structure. To address the effects of MeCP2 deficiency on heterochromatin organization during neural differentiation, we developed a versatile model for stem cell in vitro differentiation. Therefore, we modified murine Mecp2 deficient (Mecp2(-/y)) embryonic stem cells to generate cells exhibiting green fluorescent protein expression upon neural differentiation. Subsequently, we quantitatively analyzed heterochromatin organization during neural differentiation in wild type and in Mecp2 deficient cells. We found that MeCP2 protein levels increase significantly during neural differentiation and accumulate at constitutive heterochromatin. Statistical analysis of Mecp2 wild type neurons revealed a significant clustering of heterochromatin per nuclei with progressing differentiation. In contrast we found Mecp2 deficient neurons and astroglia cells to be significantly impaired in heterochromatin reorganization. Our results (i) introduce a new and manageable cellular model to study the molecular effects of Mecp2 deficiency, and (ii) support the view of MeCP2 as a central protein in heterochromatin architecture in maturating cells, possibly involved in stabilizing their differentiated state

MeCP2 Dependent Heterochromatin Reorganization during Neural Differentiation of a Novel <em>Mecp2</em>-Deficient Embryonic Stem Cell Reporter Line

<div><p>The X-linked <em>Mecp2</em> is a known interpreter of epigenetic information and mutated in Rett syndrome, a complex neurological disease. MeCP2 recruits HDAC complexes to chromatin thereby modulating gene expression and, importantly regulates higher order heterochromatin structure. To address the effects of MeCP2 deficiency on heterochromatin organization during neural differentiation, we developed a versatile model for stem cell <em>in vitro</em> differentiation. Therefore, we modified murine <em>Mecp2</em> deficient (<em>Mecp2</em>^−/y) embryonic stem cells to generate cells exhibiting green fluorescent protein expression upon neural differentiation. Subsequently, we quantitatively analyzed heterochromatin organization during neural differentiation in wild type and in <em>Mecp2</em> deficient cells. We found that MeCP2 protein levels increase significantly during neural differentiation and accumulate at constitutive heterochromatin. Statistical analysis of <em>Mecp2</em> wild type neurons revealed a significant clustering of heterochromatin per nuclei with progressing differentiation. In contrast we found <em>Mecp2</em> deficient neurons and astroglia cells to be significantly impaired in heterochromatin reorganization. Our results (i) introduce a new and manageable cellular model to study the molecular effects of <em>Mecp2</em> deficiency, and (ii) support the view of MeCP2 as a central protein in heterochromatin architecture in maturating cells, possibly involved in stabilizing their differentiated state.</p> </div