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1.
Proc Natl Acad Sci U S A ; 117(40): 24947-24956, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32968016

RESUMO

The acquisition of mutations plays critical roles in adaptation, evolution, senescence, and tumorigenesis. Massive genome sequencing has allowed extraction of specific features of many mutational landscapes but it remains difficult to retrospectively determine the mechanistic origin(s), selective forces, and trajectories of transient or persistent mutations and genome rearrangements. Here, we conducted a prospective reciprocal approach to inactivate 13 single or multiple evolutionary conserved genes involved in distinct genome maintenance processes and characterize de novo mutations in 274 diploid Saccharomyces cerevisiae mutation accumulation lines. This approach revealed the diversity, complexity, and ultimate uniqueness of mutational landscapes, differently composed of base substitutions, small insertions/deletions (InDels), structural variants, and/or ploidy variations. Several landscapes parallel the repertoire of mutational signatures in human cancers while others are either novel or composites of subsignatures resulting from distinct DNA damage lesions. Notably, the increase of base substitutions in the homologous recombination-deficient Rad51 mutant, specifically dependent on the Polζ translesion polymerase, yields COSMIC signature 3 observed in BRCA1/BRCA2-mutant breast cancer tumors. Furthermore, "mutome" analyses in highly polymorphic diploids and single-cell bottleneck lineages revealed a diverse spectrum of loss-of-heterozygosity (LOH) signatures characterized by interstitial and terminal chromosomal events resulting from interhomolog mitotic cross-overs. Following the appearance of heterozygous mutations, the strong stimulation of LOHs in the rad27/FEN1 and tsa1/PRDX1 backgrounds leads to fixation of homozygous mutations or their loss along the lineage. Overall, these mutomes and their trajectories provide a mechanistic framework to understand the origin and dynamics of genome variations that accumulate during clonal evolution.


Assuntos
Neoplasias da Mama/genética , Carcinogênese/genética , Mutação/genética , Saccharomyces cerevisiae/genética , Acetiltransferases/genética , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/patologia , Dano ao DNA/genética , DNA Polimerase Dirigida por DNA , Diploide , Feminino , Endonucleases Flap/genética , Genoma Fúngico/genética , Humanos , Perda de Heterozigosidade/genética , Proteínas de Membrana/genética , Peroxirredoxinas/genética , Rad51 Recombinase/genética , Proteínas de Saccharomyces cerevisiae/genética , Sequenciamento Completo do Genoma
3.
Kidney Int ; 98(4): 883-896, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32919786

RESUMO

Loss-of-function mutations in the OCRL gene, which encodes the phosphatidylinositol [PI] 4,5-bisphosphate [PI(4,5)P2] 5-phosphatase OCRL, cause defective endocytosis and proximal tubule dysfunction in Lowe syndrome and Dent disease 2. The defect is due to increased levels of PI(4,5)P2 and aberrant actin polymerization, blocking endosomal trafficking. PI 3-phosphate [PI(3)P] has been recently identified as a coactivator with PI(4,5)P2 in the actin pathway. Here, we tested the hypothesis that phosphoinositide 3-kinase (PI3K) inhibitors may rescue the endocytic defect imparted by OCRL loss, by rebalancing phosphoinositide signals to the actin machinery. The broad-range PI3K inhibitor copanlisib and class IA p110α PI3K inhibitor alpelisib reduced aberrant actin polymerization in OCRL-deficient human kidney cells in vitro. Levels of PI 3,4,5-trisphosphate, PI(4,5)P2 and PI(3)P were all reduced with alpelisib treatment, and siRNA knockdown of the PI3K catalytic subunit p110α phenocopied the actin phenotype. In a humanized OcrlY/- mouse model, alpelisib reduced endosomal actin staining while restoring stress fiber architecture and levels of megalin at the plasma membrane of proximal tubule cells, reflected by improved endocytic uptake of low molecular weight proteins in vivo. Thus, our findings support the link between phosphoinositide lipids, actin polymerization and endocytic trafficking in the proximal tubule and represent a proof-of-concept for repurposing alpelisib in Lowe syndrome/Dent disease 2.

4.
Cardiol Ther ; 9(2): 363-376, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32862327

RESUMO

All heart muscle diseases that cause chronic heart failure finally converge into one dreaded pathological process that is myocardial fibrosis. Myocardial fibrosis predicts major adverse cardiovascular events and death, yet we are still missing the targeted therapies capable of halting and/or reversing its progression. Fundamentally it is a problem of disproportionate extracellular collagen accumulation that is part of normal myocardial ageing and accentuated in certain disease states. In this article we discuss the role of cardiovascular magnetic resonance (CMR) imaging biomarkers to track fibrosis and collate results from the most promising animal and human trials of anti-fibrotic therapies to date. We underscore the ever-growing role of CMR in determining the efficacy of such drugs and encourage future trialists to turn to CMR when designing their surrogate study endpoints.

5.
Structure ; 28(6): 674-689.e11, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32375023

RESUMO

Centrioles are cylindrical assemblies whose peripheral microtubule array displays a 9-fold rotational symmetry that is established by the scaffolding protein SAS6. Centriole symmetry can be broken by centriole-associated structures, such as the striated fibers in Chlamydomonas that are important for ciliary function. The conserved protein CCDC61/VFL3 is involved in this process, but its exact role is unclear. Here, we show that CCDC61 is a paralog of SAS6. Crystal structures of CCDC61 demonstrate that it contains two homodimerization interfaces that are similar to those found in SAS6, but result in the formation of linear filaments rather than rings. Furthermore, we show that CCDC61 binds microtubules and that residues involved in CCDC61 microtubule binding are important for ciliary function in Chlamydomonas. Together, our findings suggest that CCDC61 and SAS6 functionally diverged from a common ancestor while retaining the ability to scaffold the assembly of basal body-associated structures or centrioles, respectively.

6.
Elife ; 92020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32441252

RESUMO

CRISPR-Cas9 genome engineering has revolutionised high-throughput functional genomic screens. However, recent work has raised concerns regarding the performance of CRISPR-Cas9 screens using TP53 wild-type human cells due to a p53-mediated DNA damage response (DDR) limiting the efficiency of generating viable edited cells. To directly assess the impact of cellular p53 status on CRISPR-Cas9 screen performance, we carried out parallel CRISPR-Cas9 screens in wild-type and TP53 knockout human retinal pigment epithelial cells using a focused dual guide RNA library targeting 852 DDR-associated genes. Our work demonstrates that although functional p53 status negatively affects identification of significantly depleted genes, optimal screen design can nevertheless enable robust screen performance. Through analysis of our own and published screen data, we highlight key factors for successful screens in both wild-type and p53-deficient cells.

7.
Nat Commun ; 11(1): 819, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-32041954

RESUMO

Loss of functional BRCA1 protein leads to defects in DNA double-strand break (DSB) repair by homologous recombination (HR) and renders cells hypersensitive to poly (ADP-ribose) polymerase (PARP) inhibitors used to treat BRCA1/2-deficient cancers. However, upon chronic treatment of BRCA1-mutant cells with PARP inhibitors, resistant clones can arise via several mechanisms, including loss of 53BP1 or its downstream co-factors. Defects in the 53BP1 axis partially restore the ability of a BRCA1-deficient cell to form RAD51 filaments at resected DSBs in a PALB2- and BRCA2-dependent manner, and thereby repair DSBs by HR. Here we show that depleting 53BP1 in BRCA1-null cells restores PALB2 accrual at resected DSBs. Moreover, we demonstrate that PALB2 DSB recruitment in BRCA1/53BP1-deficient cells is mediated by an interaction between PALB2's chromatin associated motif (ChAM) and the nucleosome acidic patch region, which in 53BP1-expressing cells is bound by 53BP1's ubiquitin-directed recruitment (UDR) domain.


Assuntos
Proteína BRCA1/deficiência , Cromatina/metabolismo , Proteína do Grupo de Complementação N da Anemia de Fanconi/metabolismo , Recombinação Homóloga , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/deficiência , Motivos de Aminoácidos , Proteína BRCA2/deficiência , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Proteína do Grupo de Complementação N da Anemia de Fanconi/química , Proteína do Grupo de Complementação N da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Humanos , Nucleossomos/metabolismo
8.
Sci Rep ; 10(1): 2200, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-32042076

RESUMO

Over the past decades, there have been huge advances in understanding cellular responses to ionising radiation (IR) and DNA damage. These studies, however, were mostly executed with cell lines and mice using single or multiple acute doses of radiation. Hence, relatively little is known about how continuous exposure to low dose ionising radiation affects normal cells and organisms, even though our cells are constantly exposed to low levels of radiation. We addressed this issue by examining the consequences of exposing human primary cells to continuous ionising γ-radiation delivered at 6-20 mGy/h. Although these dose rates are estimated to inflict fewer than a single DNA double-strand break (DSB) per hour per cell, they still caused dose-dependent reductions in cell proliferation and increased cellular senescence. We concomitantly observed histone protein levels to reduce by up to 40%, which in contrast to previous observations, was not mainly due to protein degradation but instead correlated with reduced histone gene expression. Histone reductions were accompanied by enlarged nuclear size paralleled by an increase in global transcription, including that of pro-inflammatory genes. Thus, chronic irradiation, even at low dose-rates, can induce cell senescence and alter gene expression via a hitherto uncharacterised epigenetic route. These features of chronic radiation represent a new aspect of radiation biology.


Assuntos
Cromatina/efeitos da radiação , Expressão Gênica/efeitos da radiação , Histonas/efeitos da radiação , Animais , Linhagem Celular , Proliferação de Células/efeitos da radiação , Senescência Celular/efeitos da radiação , DNA/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Dano ao DNA/efeitos da radiação , Reparo do DNA/fisiologia , Reparo do DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Raios gama , Histonas/genética , Humanos , Masculino , Camundongos , Cultura Primária de Células
9.
Nat Commun ; 10(1): 5191, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31729360

RESUMO

Histone H2AX and MDC1 are key DNA repair and DNA-damage signalling proteins. When DNA double-strand breaks (DSBs) occur, H2AX is phosphorylated and then recruits MDC1, which in turn serves as a docking platform to promote the localization of other factors, including 53BP1, to DSB sites. Here, by using CRISPR-Cas9 engineered human cell lines, we identify a hitherto unknown, H2AX-independent, function of MDC1 mediated by its PST-repeat region. We show that the PST-repeat region directly interacts with chromatin via the nucleosome acidic patch and mediates DNA damage-independent association of MDC1 with chromatin. We find that this region is largely functionally dispensable when the canonical γH2AX-MDC1 pathway is operative but becomes critical for 53BP1 recruitment to DNA-damage sites and cell survival following DSB induction when H2AX is not available. Consequently, our results suggest a role for MDC1 in activating the DDR in areas of the genome lacking or depleted of H2AX.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Dano ao DNA , Histonas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Motivos de Aminoácidos , Proteínas de Ciclo Celular/genética , Linhagem Celular , Cromatina/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Histonas/genética , Humanos , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
10.
Nature ; 573(7774): 416-420, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31511699

RESUMO

Despite major progress in defining the functional roles of genes, a complete understanding of their influences is far from being realized, even in relatively simple organisms. A major milestone in this direction arose via the completion of the yeast Saccharomyces cerevisiae gene-knockout collection (YKOC), which has enabled high-throughput reverse genetics, phenotypic screenings and analyses of synthetic-genetic interactions1-3. Ensuing experimental work has also highlighted some inconsistencies and mistakes in the YKOC, or genome instability events that rebalance the effects of specific knockouts4-6, but a complete overview of these is lacking. The identification and analysis of genes that are required for maintaining genomic stability have traditionally relied on reporter assays and on the study of deletions of individual genes, but whole-genome-sequencing technologies now enable-in principle-the direct observation of genome instability globally and at scale. To exploit this opportunity, we sequenced the whole genomes of nearly all of the 4,732 strains comprising the homozygous diploid YKOC. Here, by extracting information on copy-number variation of tandem and interspersed repetitive DNA elements, we describe-for almost every single non-essential gene-the genomic alterations that are induced by its loss. Analysis of this dataset reveals genes that affect the maintenance of various genomic elements, highlights cross-talks between nuclear and mitochondrial genome stability, and shows how strains have genetically adapted to life in the absence of individual non-essential genes.


Assuntos
Genoma Fúngico/genética , Instabilidade Genômica , Saccharomyces cerevisiae/genética , Adaptação Biológica/genética , Técnicas de Inativação de Genes , Genoma Mitocondrial/genética , Sequenciamento Completo do Genoma
11.
ACS Chem Biol ; 14(10): 2148-2154, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31525021

RESUMO

The Fanconi anemia pathway orchestrates the repair of DNA interstrand cross-links and stalled replication forks. A key step in this pathway is UBE2T and FANCL-dependent monoubiquitylation of the FANCD2-FANCI complex. The Fanconi anemia pathway represents an attractive therapeutic target, because activation of this pathway has been linked to chemotherapy resistance in several cancers. However, to date, very few selective inhibitors of ubiquitin conjugation pathways are known. By using a high-throughput screen-compatible assay, we have identified a small-molecule inhibitor of UBE2T/FANCL-mediated FANCD2 monoubiquitylation that sensitizes cells to the DNA cross-linking agent, carboplatin.


Assuntos
Proteína do Grupo de Complementação L da Anemia de Fanconi/antagonistas & inibidores , Anemia de Fanconi/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Linhagem Celular Tumoral , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação
12.
Nat Protoc ; 14(7): 1991-2014, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31160788

RESUMO

Ploidy represents the number of chromosome sets in a cell. Although gametes have a haploid genome (n), most mammalian cells have diploid genomes (2n). The diploid status of most cells correlates with the number of probable alleles for each autosomal gene and makes it difficult to target these genes via mutagenesis techniques. Here, we describe a 7-week protocol for the derivation of mouse haploid embryonic stem cells (hESCs) from female gametes that also outlines how to maintain the cells once derived. We detail additional procedures that can be used with cell lines obtained from the mouse Haplobank, a biobank of >100,000 individual mouse hESC lines with targeted mutations in 16,970 genes. hESCs can spontaneously diploidize and can be maintained in both haploid and diploid states. Mouse hESCs are genomically and karyotypically stable, are innately immortal and isogenic, and can be derived in an array of differentiated cell types; they are thus highly amenable to genetic screens and to defining molecular connectivity pathways.


Assuntos
Técnicas de Cultura de Células/métodos , Haploidia , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/fisiologia , Animais , Blastocisto/citologia , Linhagem Celular , Separação Celular/métodos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Fluxo de Trabalho
13.
Science ; 364(6437): 247, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30975768
14.
Nature ; 568(7753): 576, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30976101

RESUMO

This Article has been retracted; see accompanying Retraction.

15.
Cell ; 177(4): 821-836.e16, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30982602

RESUMO

Whole-genome-sequencing (WGS) of human tumors has revealed distinct mutation patterns that hint at the causative origins of cancer. We examined mutational signatures in 324 WGS human-induced pluripotent stem cells exposed to 79 known or suspected environmental carcinogens. Forty-one yielded characteristic substitution mutational signatures. Some were similar to signatures found in human tumors. Additionally, six agents produced double-substitution signatures and eight produced indel signatures. Investigating mutation asymmetries across genome topography revealed fully functional mismatch and transcription-coupled repair pathways. DNA damage induced by environmental mutagens can be resolved by disparate repair and/or replicative pathways, resulting in an assortment of signature outcomes even for a single agent. This compendium of experimentally induced mutational signatures permits further exploration of roles of environmental agents in cancer etiology and underscores how human stem cell DNA is directly vulnerable to environmental agents. VIDEO ABSTRACT.


Assuntos
Carcinógenos Ambientais/classificação , Neoplasias/genética , Carcinógenos Ambientais/efeitos adversos , Dano ao DNA/genética , Análise Mutacional de DNA/métodos , Reparo do DNA/genética , Replicação do DNA , Perfil Genético , Genoma Humano/genética , Humanos , Mutação INDEL/genética , Mutagênese , Mutação/genética , Células-Tronco Pluripotentes/metabolismo , Sequenciamento Completo do Genoma/métodos
16.
Nat Commun ; 10(1): 87, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30622252

RESUMO

Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase I inhibitor topotecan. Thus, we here establish that inactivating terminal components of the non-homologous end-joining (NHEJ) machinery or of the BRCA1-A complex specifically confer topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase (PARP) inhibitor olaparib reflects delayed engagement of homologous recombination at DNA-replication-fork associated single-ended double-strand breaks (DSBs), allowing some to be subject to toxic NHEJ. Preventing DSB ligation by NHEJ, or enhancing homologous recombination by BRCA1-A complex disruption, suppresses this toxicity, highlighting a crucial role for ATM in preventing toxic LIG4-mediated chromosome fusions. Notably, suppressor mutations in ATM-mutant backgrounds are different to those in BRCA1-mutant scenarios, suggesting new opportunities for patient stratification and additional therapeutic vulnerabilities for clinical exploitation.


Assuntos
Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Reparo do DNA por Junção de Extremidades/genética , Resistencia a Medicamentos Antineoplásicos/genética , Animais , Antineoplásicos/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Ligase Dependente de ATP/metabolismo , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Células-Tronco Embrionárias Murinas , Mutação , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Ftalazinas/farmacologia , Ftalazinas/uso terapêutico , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Topotecan/farmacologia , Topotecan/uso terapêutico
17.
Cell Rep ; 26(3): 582-593.e5, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30650353

RESUMO

The neuronal microtubule-associated protein tau, MAPT, is central to the pathogenesis of many dementias. Autosomal-dominant mutations in MAPT cause inherited frontotemporal dementia (FTD), but the underlying pathogenic mechanisms are unclear. Using human stem cell models of FTD due to MAPT mutations, we find that tau becomes hyperphosphorylated and mislocalizes to cell bodies and dendrites in cortical neurons, recapitulating a key early event in FTD. Mislocalized tau in the cell body leads to abnormal microtubule movements in FTD-MAPT neurons that grossly deform the nuclear membrane. This results in defective nucleocytoplasmic transport, which is corrected by microtubule depolymerization. Neurons in the post-mortem human FTD-MAPT cortex have a high incidence of nuclear invaginations, indicating that tau-mediated nuclear membrane dysfunction is an important pathogenic process in FTD. Defects in nucleocytoplasmic transport in FTD point to important commonalities in the pathogenic mechanisms of tau-mediated dementias and ALS-FTD due to TDP-43 and C9orf72 mutations.


Assuntos
Transporte Ativo do Núcleo Celular/genética , Demência Frontotemporal/genética , Microtúbulos/metabolismo , Membrana Nuclear/metabolismo , Demência Frontotemporal/patologia , Humanos
18.
Nat Biotechnol ; 2018 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-30480667

RESUMO

The DNA mutation produced by cellular repair of a CRISPR-Cas9-generated double-strand break determines its phenotypic effect. It is known that the mutational outcomes are not random, but depend on DNA sequence at the targeted location. Here we systematically study the influence of flanking DNA sequence on repair outcome by measuring the edits generated by >40,000 guide RNAs (gRNAs) in synthetic constructs. We performed the experiments in a range of genetic backgrounds and using alternative CRISPR-Cas9 reagents. In total, we gathered data for >109 mutational outcomes. The majority of reproducible mutations are insertions of a single base, short deletions or longer microhomology-mediated deletions. Each gRNA has an individual cell-line-dependent bias toward particular outcomes. We uncover sequence determinants of the mutations produced and use these to derive a predictor of Cas9 editing outcomes. Improved understanding of sequence repair will allow better design of gene editing experiments.

19.
Mol Cell ; 72(4): 625-635.e4, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30454561

RESUMO

In response to genotoxic stress, cells activate a signaling cascade known as the DNA damage checkpoint (DDC) that leads to a temporary cell cycle arrest and activation of DNA repair mechanisms. Because persistent DDC activation compromises cell viability, this process must be tightly regulated. However, despite its importance, the mechanisms regulating DDC recovery are not completely understood. Here, we identify a DNA-damage-regulated histone modification in Saccharomyces cerevisiae, phosphorylation of H4 threonine 80 (H4T80ph), and show that it triggers checkpoint inactivation. H4T80ph is critical for cell survival to DNA damage, and its absence causes impaired DDC recovery and persistent cell cycle arrest. We show that, in response to genotoxic stress, p21-activated kinase Cla4 phosphorylates H4T80 to recruit Rtt107 to sites of DNA damage. Rtt107 displaces the checkpoint adaptor Rad9, thereby interrupting the checkpoint-signaling cascade. Collectively, our results indicate that H4T80ph regulates DDC recovery.


Assuntos
Dano ao DNA , Reparo do DNA , Histonas/genética , Histonas/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais
20.
Sci Signal ; 11(537)2018 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-29970603

RESUMO

Hutchinson-Gilford progeria syndrome (HGPS) is an incurable premature aging disease. Identifying deregulated biological processes in HGPS might thus help define novel therapeutic strategies. Fibroblasts from HGPS patients display defects in nucleocytoplasmic shuttling of the GTP-bound form of the small GTPase Ran (RanGTP), which leads to abnormal transport of proteins into the nucleus. We report that microtubule stabilization in HGPS cells sequestered the nonclassical nuclear import protein Transportin-1 (TNPO1) in the cytoplasm, thus affecting the nuclear localization of its cargo, including the nuclear pore protein NUP153. Consequently, nuclear Ran, nuclear anchorage of the nucleoporin TPR, and chromatin organization were disrupted, deregulating gene expression and inducing senescence. Inhibiting N-acetyltransferase 10 (NAT10) ameliorated HGPS phenotypes by rebalancing the nuclear to cytoplasmic ratio of TNPO1. This restored nuclear pore complex integrity and nuclear Ran localization, thereby correcting HGPS cellular phenotypes. We observed a similar mechanism in cells from healthy aged individuals. This study identifies a nuclear import pathway affected in aging and underscores the potential for NAT10 inhibition as a possible therapeutic strategy for HGPS and perhaps also for pathologies associated with normal aging.


Assuntos
Núcleo Celular/metabolismo , Senescência Celular , Acetiltransferase N-Terminal E/antagonistas & inibidores , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Progéria/prevenção & controle , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Adulto , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Células Cultivadas , Criança , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Masculino , Microtúbulos/metabolismo , Microtúbulos/patologia , Acetiltransferase N-Terminal E/genética , Acetiltransferase N-Terminal E/metabolismo , Acetiltransferases N-Terminal , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Fenótipo , Progéria/genética , Progéria/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Adulto Jovem , beta Carioferinas/genética , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismo
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