RESUMO
Werner syndrome (WS) is an autosomal recessive disease caused by loss of function of WRN. WS is a segmental progeroid disease and shows early onset or increased frequency of many characteristics of normal aging. WRN possesses helicase, annealing, strand exchange, and exonuclease activities and acts on a variety of DNA substrates, even complex replication and recombination intermediates. Here, we review the genetics, biochemistry, and probably physiological functions of the WRN protein. Although its precise role is unclear, evidence suggests WRN plays a role in pathways that respond to replication stress and maintain genome stability particularly in telomeric regions.
Assuntos
Replicação do DNA , Instabilidade Genômica , Helicase da Síndrome de Werner , Síndrome de Werner , Helicase da Síndrome de Werner/metabolismo , Helicase da Síndrome de Werner/genética , Humanos , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Animais , Telômero/metabolismo , Telômero/genéticaRESUMO
The underlying mechanisms of atherosclerosis, the second leading cause of death among Werner syndrome (WS) patients, are not fully understood. Here, we establish an in vitro co-culture system using macrophages (iMφs), vascular endothelial cells (iVECs), and vascular smooth muscle cells (iVSMCs) derived from induced pluripotent stem cells. In co-culture, WS-iMφs induces endothelial dysfunction in WS-iVECs and characteristics of the synthetic phenotype in WS-iVSMCs. Transcriptomics and open chromatin analysis reveal accelerated activation of type I interferon signaling and reduced chromatin accessibility of several transcriptional binding sites required for cellular homeostasis in WS-iMφs. Furthermore, the H3K9me3 levels show an inverse correlation with retrotransposable elements, and retrotransposable element-derived double-stranded RNA activates the DExH-box helicase 58 (DHX58)-dependent cytoplasmic RNA sensing pathway in WS-iMφs. Conversely, silencing type I interferon signaling in WS-iMφs rescues cell proliferation and suppresses cellular senescence and inflammation. These findings suggest that Mφ-specific inhibition of type I interferon signaling could be targeted to treat atherosclerosis in WS patients.
Assuntos
Aterosclerose , Inflamação , Interferon Tipo I , Macrófagos , Retroelementos , Síndrome de Werner , Interferon Tipo I/metabolismo , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Humanos , Aterosclerose/metabolismo , Aterosclerose/imunologia , Aterosclerose/genética , Aterosclerose/patologia , Macrófagos/metabolismo , Macrófagos/imunologia , Retroelementos/genética , Inflamação/metabolismo , Inflamação/patologia , Inflamação/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Transdução de Sinais , Técnicas de Cocultura , Miócitos de Músculo Liso/metabolismo , Células Endoteliais/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Senescência Celular , Proliferação de CélulasRESUMO
Interstitial lung disease (ILD) is a condition affecting the lung parenchyma by inflammation and fibrosis and can be caused by various exposures, connective tissue diseases (CTD), and genetic disorders. In this report, a family with five patients having progressive respiratory failure that begins with coughing in adolescence, followed by dyspnea and recurrent spontaneous pneumothorax, and death in early adulthood is presented. The patients were diagnosed to have ILD through clinical and radiological evaluations. Molecular genetic analyses of the family provided two homozygous rare variants in the WRN and SFXN5 genes, co-segregating with the phenotype. The network analyses pointed out that the variant in the WRN, rather than that in the SFXN5 gene, could be the main factor in the existence of the ILD phenotype, putatively through the altered DNA repair and telomere maintenance pathways. In silico analyses suggested that the variant could affect the exonuclease activity or the stability of the WRN protein. Moreover, the adolescent-onset pulmonary phenotype described in the case has not been reported in Werner Syndrome, the only disease known to be associated with biallelic WRN pathogenic variants. Thus, the present phenotype could be either a very atypical presentation of Werner syndrome or a new clinical entity associated with the WRN gene.
Assuntos
Doenças Pulmonares Intersticiais , Pneumotórax , Síndrome de Werner , Humanos , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Doenças Pulmonares Intersticiais/complicações , Doenças Pulmonares Intersticiais/diagnóstico , Doenças Pulmonares Intersticiais/genética , Pneumotórax/diagnóstico , Pneumotórax/genética , RecQ Helicases/genética , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Síndrome de Werner/patologia , Helicase da Síndrome de Werner/genética , Helicase da Síndrome de Werner/metabolismoRESUMO
Hereditary breast and ovarian cancers are frequently attributed to germline mutations in the tumor suppressor genes BRCA1 and BRCA2. BRCA1/2 act to repair double-strand breaks (DSBs) and suppress the demise of unstable replication forks. Our work elucidated a dynamic interplay between BRCA2 and the WRN DNA helicase/exonuclease defective in the premature aging disorder Werner syndrome. WRN and BRCA2 participate in complementary pathways to stabilize replication forks in cancer cells, allowing them to proliferate. Whether the functional overlap of WRN and BRCA2 is relevant to replication at gaps between newly synthesized DNA fragments, protection of telomeres, and/or metabolism of secondary DNA structures remain to be determined. Advances in understanding the mechanisms elicited during replication stress have prompted the community to reconsider avenues for cancer therapy. Insights from studies of PARP or topoisomerase inhibitors provide working models for the investigation of WRN's mechanism of action. We discuss these topics, focusing on the implications of the WRN-BRCA2 genetic interaction under conditions of replication stress.
Assuntos
Senilidade Prematura , Replicação do DNA , Neoplasias , Síndrome de Werner , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Instabilidade Cromossômica , DNA Helicases/química , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Helicase da Síndrome de Werner/genética , Helicase da Síndrome de Werner/metabolismoRESUMO
Oxidatively damaged bases induce mutations and are involved in cancer initiation. 8-Oxo-7,8-dihydroguanine (G°, 8-hydroxyguanine) is an abundant oxidized base that induces targeted G:CâT:A transversions in human cells, as well as untargeted base substitution (action-at-a-distance) mutations of the G bases of 5'-GpA-3' dinucleotides. The action-at-a-distance mutations become more frequent than the targeted transversions when the amount of Werner syndrome (WRN) protein is decreased. In this study, OGG1, the major DNA glycosylase for the damaged base, and WRN were knocked down in isolation and in combination in human U2OS cells, and a shuttle plasmid carrying G° was introduced into the knockdown cells. Interestingly, fewer action-at-a-distance mutations were observed in the WRN plus OGG1 double knockdown cells, as compared to the WRN single knockdown cells. These results indicated the paradoxical role of OGG1, as an accelerator of the action-at-a-distance mutations by the oxidized guanine base.
Assuntos
DNA Glicosilases/metabolismo , Síndrome de Werner , Dano ao DNA , DNA Glicosilases/genética , Reparo do DNA , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Mutação , Síndrome de Werner/metabolismoRESUMO
Werner syndrome (WS) is an extremely rare, autosomal recessive segmental progeroid disorder caused by biallelic pathogenic variants in the WRN, which encodes a multifunctional nuclear protein that belongs to the RecQ family of DNA helicases. Despite extensive research on WS in the last years, the population-specific mutational spectrum still needs to be elucidated. Moreover, there is an evident lack of detailed clinical descriptions accompanied with photographs of affected individuals. Here, we report a consanguineous Lebanese family in whom we identified a pathogenic homozygous nonsense variant c.1111G>T, p.Glu371* in the WRN. The index individual, at the age of 54 years, was suspected to have WS due to a history of early-onset cataracts, premature hair loss and graying, chronic nonhealing leg ulcers, Achilles' tendon calcifications, type 2 diabetes mellitus, dyslipidemia, hypothyroidism, and premature coronary artery disease. His four sisters, three of which deceased in the fifth decade, had clinical signs suggestive of WS. Moreover, his daughter, aged 23 years, had short stature, hair loss and flat feet. Taken together, we report a detailed clinical course of disease in several affected members of a consanguineous family, which is additionally documented by photographs.
Assuntos
Diabetes Mellitus Tipo 2 , Síndrome de Werner , Alopecia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RecQ Helicases/genética , Síndrome de Werner/diagnóstico , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Helicase da Síndrome de Werner/genética , Helicase da Síndrome de Werner/metabolismo , Adulto JovemRESUMO
Werner syndrome (WS) is an accelerated aging disorder characterized by genomic instability, which is caused by WRN protein deficiency. WRN participates in DNA metabolism including DNA repair. In a previous report, we showed that WRN protein is recruited to laser-induced DNA double-strand break (DSB) sites during various stages of the cell cycle with similar intensities, supporting that WRN participates in both non-homologous end joining (NHEJ) and homologous recombination (HR). Here, we demonstrate that the phosphorylation of WRN by CDK2 on serine residue 426 is critical for WRN to make its DSB repair pathway choice between NHEJ and HR. Cells expressing WRN engineered to mimic the unphosphorylated or phosphorylation state at serine 426 showed abnormal DSB recruitment, altered RPA interaction, strand annealing, and DSB repair activities. The CDK2 phosphorylation on serine 426 stabilizes WRN's affinity for RPA, likely increasing its long-range resection at the end of DNA strands, which is a crucial step for HR. Collectively, the data shown here demonstrate that a CDK2-dependent phosphorylation of WRN regulates DSB repair pathway choice and cell cycle participation.
Assuntos
Quinase 2 Dependente de Ciclina/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA por Junção de Extremidades/genética , Recombinação Homóloga , Transdução de Sinais/genética , Helicase da Síndrome de Werner/metabolismo , Ciclo Celular/genética , Linhagem Celular Tumoral , Quinase 2 Dependente de Ciclina/genética , DNA/metabolismo , Células HEK293 , Humanos , Fosforilação/genética , Proteína de Replicação A/metabolismo , Serina/metabolismo , Transfecção , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Helicase da Síndrome de Werner/genéticaRESUMO
Werner syndrome (WS) is an accelerated ageing disease caused by multiple mutations in the gene encoding the Werner DNA helicase (WRN). The major clinical features of WS include wrinkles, grey hair, osteoporosis, and metabolic phenomena such as atherosclerosis, diabetes, and fatty liver, and resemble those seen in normal ageing, but occur earlier, in middle age. Defective DNA repair resulting from mutations in WRN explain the majority of the clinical features of WS, but the underlying mechanisms driving the larger metabolic dysfunction remain elusive. Recent studies in animal models of WS and in WS patient cells and blood samples suggest the involvement of impaired mitophagy, NAD+ depletion, and accumulation of damaged mitochondria in metabolic dysfunction. This mini-review summarizes recent progress in the understanding of the molecular mechanisms of metabolic dysfunction in WS, with the involvement of DNA damage, mitochondrial dysfunction, mitophagy reduction, stem cell impairment, and senescence. Future studies on NAD+ and mitophagy may shed light on potential therapeutic strategies for the WS patients.
Assuntos
Envelhecimento/genética , Dano ao DNA , Mitocôndrias/genética , Mitofagia/genética , Células-Tronco/metabolismo , Síndrome de Werner/genética , Animais , Senescência Celular/genética , Humanos , Mitocôndrias/metabolismo , Telômero/genética , Telômero/metabolismo , Síndrome de Werner/metabolismo , Síndrome de Werner/patologiaRESUMO
G:C sites distant from 8-oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine) are frequently mutated when the lesion-bearing plasmid DNA is replicated in human cells with reduced Werner syndrome (WRN) protein. To detect the untargeted mutations preferentially, the oxidised guanine base was placed downstream of the reporter supF gene and the plasmid DNA was introduced into WRN-knockdown cells. The total mutant frequency seemed higher in the WRN-knockdown cells as compared to the control cells. Mutation analyses revealed that substitution mutations occurred at the G:C pairs of 5'-GpA-3'/5'-TpC-3' sites, the preferred sequence for the apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3)-family cytosine deaminases, in the supF gene in both control and knockdown cells. These mutations were observed more frequently at G sites than C sites on the DNA strand where the GO base was originally located. This tendency was promoted by the knockdown of the WRN protein. The present results imply the possible involvement of APOBEC3-family cytosine deaminases in the action-at-a-distance (untargeted) mutations at G:C (or G) sites induced by GO and in cancer initiation by oxidative stress.
Assuntos
Guanina , Mutação , Helicase da Síndrome de Werner/genética , Síndrome de Werner/genética , Sequência de Bases , Linhagem Celular , Técnicas de Silenciamento de Genes , Ordem dos Genes , Guanina/metabolismo , Humanos , Taxa de Mutação , Plasmídeos/genética , Síndrome de Werner/metabolismo , Helicase da Síndrome de Werner/metabolismoRESUMO
Caenorhabditis elegans expresses human Werner syndrome protein (WRN) orthologs as two distinct proteins: MUT-7, with a 3'-5' exonuclease domain, and CeWRN-1, with helicase domains. How these domains cooperate remains unclear. Here, we demonstrate the different contributions of MUT-7 and CeWRN-1 to 22G small interfering RNA (siRNA) synthesis and the plasticity of neuronal signaling. MUT-7 acts specifically in the cytoplasm to promote siRNA biogenesis and in the nucleus to associate with CeWRN-1. The import of siRNA by the nuclear Argonaute NRDE-3 promotes the loading of the heterochromatin-binding protein HP1 homolog HPL-2 onto specific loci. This heterochromatin complex represses the gene expression of the guanylyl cyclase ODR-1 to direct olfactory plasticity in C. elegans. Our findings suggest that the exonuclease and helicase domains of human WRN may act in concert to promote RNA-dependent loading into a heterochromatin complex, and the failure of this entire process reduces plasticity in postmitotic neurons.
Assuntos
DNA Helicases/metabolismo , Helicase da Síndrome de Werner/metabolismo , Síndrome de Werner/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , DNA Helicases/genética , Exonucleases , Heterocromatina , Plasticidade Neuronal , RNA Interferente Pequeno/metabolismo , Síndrome de Werner/genética , Helicase da Síndrome de Werner/genéticaRESUMO
Werner syndrome, also called adult progeria, is a heritable autosomal recessive human disorder characterized by the premature onset of numerous age-related diseases including juvenile cataracts, dyslipidemia, diabetes mellitus (DM), osteoporosis, atherosclerosis, and cancer. Werner syndrome is a segmental progeroid syndrome whose presentation resembles accelerated aging. The most common causes of death for WS patients are atherosclerosis and cancer. A 40-year-old female presented with short stature, bird-like facies, canities with alopecia, scleroderma-like skin changes, and non-healing foot ulcers. The patient reported a history of delayed puberty, abortion, hypertriglyceridemia, and juvenile cataracts. A clinical diagnosis of WS was made and subsequently confirmed. We discovered two WRN gene mutations in the patient, Variant 1 was the most common WRN mutation, nonsense mutation (c.1105C>T:p.R369Ter) in exon 9, which caused a premature termination codon (PTC) at position 369. Variant 2 was a frameshift mutation (c.1134delA:p.E379KfsTer5) in exon 9, which caused a PTC at position 383 and has no published reports describing. Patients with WS can show a wide variety of clinical and biological manifestations in endocrine-metabolic systems (DM, thyroid dysfunction, and hyperlipidemia). Doctors must be cognizant of early manifestations of WS and treatment options.
Assuntos
Doenças Ósseas Metabólicas/fisiopatologia , Diabetes Mellitus Tipo 2/metabolismo , Fígado Gorduroso/fisiopatologia , Hipertrigliceridemia/metabolismo , Hipotireoidismo/metabolismo , Síndrome de Werner/metabolismo , Aborto Habitual/fisiopatologia , Tecido Adiposo/diagnóstico por imagem , Adulto , Alopecia/fisiopatologia , Composição Corporal , Doenças Ósseas Metabólicas/diagnóstico por imagem , Catarata/fisiopatologia , Códon sem Sentido , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/fisiopatologia , Pé Diabético/etiologia , Pé Diabético/fisiopatologia , Fígado Gorduroso/diagnóstico por imagem , Feminino , Mutação da Fase de Leitura , Humanos , Hipotireoidismo/fisiopatologia , Gordura Intra-Abdominal/diagnóstico por imagem , Útero/anormalidades , Síndrome de Werner/diagnóstico , Síndrome de Werner/genética , Síndrome de Werner/fisiopatologia , Helicase da Síndrome de Werner/genéticaRESUMO
INTRODUCTION: Werner protein (WRN) plays an important role in DNA repair, replication, transcription, and consequently genomic stability via its DNA-helicase and exonuclease activity. Loss of function of WRN is associated with Werner syndrome (WS), which is characterized by premature aging and cancer predisposition. Malignancies that are commonly linked to WS are thyroid carcinoma, melanoma, breast cancer, meningioma, and soft tissue and bone sarcomas. Currently, the clinicopathologic significance of WRN in breast cancer is largely unknown. PATIENTS AND METHODS: We investigated the clinicopathologic and prognostic significance of WRN protein expression in a cohort of clinically annotated series of sporadic (n = 1650) and BRCA-mutated (n = 75) invasive breast cancers. We correlated WRN protein expression to clinicopathologic characteristics, DNA repair protein expression, and survival outcomes. RESULTS: There is strong evidence of association between low nuclear and cytoplasmic WRN co-expression and low levels of KU70/KU80, DNA-PK, DNA Pol-B, CKD18, cytoplasmic RECQL4, and nuclear BLM protein expression (adjusted P-values < .05). Tumors with low nuclear or cytoplasmic WRN expression have worse overall breast cancer-specific survival (BCSS) (adjusted P-values < .05). In topoisomerase I overexpressed tumors, low WRN nuclear expression was associated with poor BCSS (P-value < .05). In BRCA-mutated tumors, low WRN cytoplasmic expression conferred shortest BCSS (P < .05). CONCLUSIONS: Low WRN protein expression is associated with poor BCSS in patients with breast cancer. This can be used to optimize the risk stratification for personalized treatment.
Assuntos
Neoplasias da Mama/metabolismo , Helicase da Síndrome de Werner/metabolismo , Síndrome de Werner/metabolismo , Senilidade Prematura/metabolismo , Neoplasias da Mama/complicações , Feminino , Humanos , Síndrome de Werner/complicaçõesRESUMO
Significance: Werner syndrome (WS) is a rare autosomal recessive malady typified by a pro-oxidant/proinflammatory status, genetic instability, and by the early onset of numerous age-associated illnesses. The protein malfunctioning in WS individuals (WRN) is a helicase/exonuclease implicated in transcription, DNA replication/repair, and telomere maintenance. Recent Advances: In the last two decades, a series of important biological systems were created to comprehend at the molecular level the effect of a defective WRN protein. Such biological tools include mouse and worm (Caenorhabditis elegans) with a mutation in the Wrn helicase ortholog as well as human WS-induced pluripotent stem cells that can ultimately be differentiated into most cell lineages. Such WS models have identified anomalies related to the hallmarks of aging. Most importantly, vitamin C counteracts these age-related cellular phenotypes in these systems. Critical Issues: Vitamin C is the only antioxidant agent capable of reversing the cellular aging-related phenotypes in those biological systems. Since vitamin C is a cofactor for many hydroxylases and mono- or dioxygenase, it adds another level of complexity in deciphering the exact molecular pathways affected by this vitamin. Moreover, it is still unclear whether a short- or long-term vitamin C supplementation in human WS patients who already display aging-related phenotypes will have a beneficial impact. Future Directions: The discovery of new molecular markers specific to the modified biological pathways in WS that can be used for novel imaging techniques or as blood markers will be necessary to assess the favorable effect of vitamin C supplementation in WS. Antioxid. Redox Signal. 34, 856-874.
Assuntos
Ácido Ascórbico/uso terapêutico , Helicase da Síndrome de Werner/genética , Síndrome de Werner/dietoterapia , Animais , Ácido Ascórbico/genética , Ácido Ascórbico/metabolismo , Caenorhabditis elegans/genética , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Suplementos Nutricionais , Modelos Animais de Doenças , Humanos , Camundongos , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Síndrome de Werner/patologiaRESUMO
BACKGROUND: The Werner syndrome protein (WRN) belongs to the RecQ family of helicases and its loss of function results in the premature aging disease Werner syndrome (WS). We previously demonstrated that an early cellular change induced by WRN depletion is a posttranscriptional decrease in the levels of enzymes involved in metabolic pathways that control macromolecular synthesis and protect from oxidative stress. This metabolic shift is tolerated by normal cells but causes mitochondria dysfunction and acute oxidative stress in rapidly growing cancer cells, thereby suppressing their proliferation. RESULTS: To identify the mechanism underlying this metabolic shift, we examined global protein synthesis and mRNA nucleocytoplasmic distribution after WRN knockdown. We determined that WRN depletion in HeLa cells attenuates global protein synthesis without affecting the level of key components of the mRNA export machinery. We further observed that WRN depletion affects the nuclear export of mRNAs and demonstrated that WRN interacts with mRNA and the Nuclear RNA Export Factor 1 (NXF1). CONCLUSIONS: Our findings suggest that WRN influences the export of mRNAs from the nucleus through its interaction with the NXF1 export receptor thereby affecting cellular proteostasis. In summary, we identified a new partner and a novel function of WRN, which is especially important for the proliferation of cancer cells.
Assuntos
Núcleo Celular/metabolismo , Neoplasias/metabolismo , RNA Mensageiro/genética , Helicase da Síndrome de Werner/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Células HeLa , Humanos , Redes e Vias Metabólicas/fisiologia , Oxirredução , Processamento Pós-Transcricional do RNA/fisiologia , Transporte de RNA/fisiologia , Proteínas de Ligação a RNA/metabolismo , RecQ Helicases/genética , Síndrome de Werner/metabolismoRESUMO
Werner Syndrome (WS) and Bloom Syndrome (BS) are disorders of DNA damage repair caused by biallelic disruption of the WRN or BLM DNA helicases respectively. Both are commonly associated with insulin resistant diabetes, usually accompanied by dyslipidemia and fatty liver, as seen in lipodystrophies. In keeping with this, progressive reduction of subcutaneous adipose tissue is commonly observed. To interrogate the underlying cause of adipose tissue dysfunction in these syndromes, CRISPR/Cas9 genome editing was used to generate human pluripotent stem cell (hPSC) lacking either functional WRN or BLM helicase. No deleterious effects were observed in WRN-/- or BLM-/- embryonic stem cells, however upon their differentiation into adipocyte precursors (AP), premature senescence emerged, impairing later stages of adipogenesis. The resulting adipocytes were also found to be senescent, with increased levels of senescent markers and senescence-associated secretory phenotype (SASP) components. SASP components initiate and reinforce senescence in adjacent cells, which is likely to create a positive feedback loop of cellular senescence within the adipocyte precursor compartment, as demonstrated in normal ageing. Such a scenario could progressively attenuate adipose mass and function, giving rise to "lipodystrophy-like" insulin resistance. Further assessment of pharmacological senolytic strategies are warranted to mitigate this component of Werner and Bloom syndromes.
Assuntos
Adipócitos/metabolismo , Síndrome de Bloom , Senescência Celular , Células-Tronco Embrionárias Humanas/metabolismo , Modelos Biológicos , Síndrome de Werner , Adipócitos/patologia , Síndrome de Bloom/genética , Síndrome de Bloom/metabolismo , Síndrome de Bloom/patologia , Sistemas CRISPR-Cas , Linhagem Celular , Deleção de Genes , Células-Tronco Embrionárias Humanas/patologia , Humanos , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Síndrome de Werner/patologiaRESUMO
WRN mutation causes a premature aging disease called Werner syndrome (WS). However, the mechanism by which WRN loss leads to progeroid features evident with impaired tissue repair and regeneration remains unclear. To determine this mechanism, we performed gene editing in reprogrammed induced pluripotent stem cells (iPSCs) derived from WS fibroblasts. Gene correction restored the expression of WRN. WRN+/+ mesenchymal stem cells (MSCs) exhibited improved pro-angiogenesis. An analysis of paracrine factors revealed that hepatocyte growth factor (HGF) was downregulated in WRN-/- MSCs. HGF insufficiency resulted in poor angiogenesis and cutaneous wound healing. Furthermore, HGF was partially regulated by PI3K/AKT signaling, which was desensitized in WRN-/- MSCs. Consistently, the inhibition of the PI3K/AKT pathway in WRN+/+ MSC resulted in reduced angiogenesis and poor wound healing. Our findings indicate that the impairment in the pro-angiogenic function of WS-MSCs is due to HGF insufficiency and PI3K/AKT dysregulation, suggesting trophic disruption between stromal and epithelial cells as a mechanism for WS pathogenesis.
Assuntos
Fator de Crescimento de Hepatócito/metabolismo , Células-Tronco Mesenquimais/metabolismo , Neovascularização Patológica/metabolismo , Helicase da Síndrome de Werner/genética , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Senescência Celular , Edição de Genes , Humanos , Células-Tronco Mesenquimais/patologia , Neovascularização Patológica/patologiaRESUMO
Metabolic dysfunction is a primary feature of Werner syndrome (WS), a human premature aging disease caused by mutations in the gene encoding the Werner (WRN) DNA helicase. WS patients exhibit severe metabolic phenotypes, but the underlying mechanisms are not understood, and whether the metabolic deficit can be targeted for therapeutic intervention has not been determined. Here we report impaired mitophagy and depletion of NAD+, a fundamental ubiquitous molecule, in WS patient samples and WS invertebrate models. WRN regulates transcription of a key NAD+ biosynthetic enzyme nicotinamide nucleotide adenylyltransferase 1 (NMNAT1). NAD+ repletion restores NAD+ metabolic profiles and improves mitochondrial quality through DCT-1 and ULK-1-dependent mitophagy. At the organismal level, NAD+ repletion remarkably extends lifespan and delays accelerated aging, including stem cell dysfunction, in Caenorhabditis elegans and Drosophila melanogaster models of WS. Our findings suggest that accelerated aging in WS is mediated by impaired mitochondrial function and mitophagy, and that bolstering cellular NAD+ levels counteracts WS phenotypes.
Assuntos
Senilidade Prematura/metabolismo , Mitofagia , NAD/metabolismo , Helicase da Síndrome de Werner/metabolismo , Síndrome de Werner/metabolismo , Senilidade Prematura/genética , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Modelos Animais de Doenças , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutação , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Síndrome de Werner/genética , Helicase da Síndrome de Werner/genéticaRESUMO
Stabilization of stalled replication forks prevents excessive fork reversal or degradation, which can undermine genome integrity. The WRN protein is unique among the other human RecQ family members to possess exonuclease activity. However, the biological role of the WRN exonuclease is poorly defined. Recently, the WRN exonuclease has been linked to protection of stalled forks from degradation. Alternative processing of perturbed forks has been associated to chemoresistance of BRCA-deficient cancer cells. Thus, we used WRN exonuclease-deficiency as a model to investigate the fate of perturbed forks undergoing degradation, but in a BRCA wild-type condition. We find that, upon treatment with clinically-relevant nanomolar doses of the Topoisomerase I inhibitor camptothecin, loss of WRN exonuclease stimulates fork inactivation and accumulation of parental gaps, which engages RAD51. Such mechanism affects reinforcement of CHK1 phosphorylation and causes persistence of RAD51 during recovery from treatment. Notably, in WRN exonuclease-deficient cells, persistence of RAD51 correlates with elevated mitotic phosphorylation of MUS81 at Ser87, which is essential to prevent excessive mitotic abnormalities. Altogether, these findings indicate that aberrant fork degradation, in the presence of a wild-type RAD51 axis, stimulates RAD51-mediated post-replicative repair and engagement of the MUS81 complex to limit genome instability and cell death.
Assuntos
Camptotecina/farmacologia , Replicação do DNA/efeitos dos fármacos , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/fisiologia , Endonucleases/fisiologia , Conformação de Ácido Nucleico/efeitos dos fármacos , Rad51 Recombinase/fisiologia , Inibidores da Topoisomerase I/farmacologia , Helicase da Síndrome de Werner/deficiência , Proteína BRCA2/fisiologia , Linhagem Celular Transformada , Quinase 1 do Ponto de Checagem/metabolismo , Quebras de DNA de Cadeia Dupla , Ativação Enzimática , Fibroblastos , Humanos , Mitocôndrias/efeitos dos fármacos , Mitose/efeitos dos fármacos , Complexos Multiproteicos/metabolismo , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Interferência de RNA , Síndrome de Werner/metabolismo , Helicase da Síndrome de Werner/fisiologiaRESUMO
MDM2 (Murine double minute 2) acts as a key repressor for p53-mediated tumor-suppressor functions, which includes cellular senescence. We found that MDM2 can promote cellular senescence by modulating WRN stability. Werner syndrome (WS), caused by mutations of the WRN gene, is an autosomal recessive disease, which is characterized by premature aging. Loss of WRN function induces cellular senescence in human cancer cells. Here, we found that MDM2 acts as an E3 ligase for WRN protein. MDM2 interacts with WRN both in vivo and in vitro. MDM2 induces ubiquitination of WRN and dramatically downregulates the levels of WRN protein in human cells. During DNA damage response, WRN is translocated to the nucleoplasm to facilitate its DNA repair functions; however, it is degraded by the MDM2-mediated ubiquitination pathway. Moreover, the senescent phenotype induced by DNA damage reagents, such as Etoposide, is at least in part mediated by MDM2-dependent WRN degradation as it can be significantly attenuated by ectopic expression of WRN. These results show that MDM2 is critically involved in regulating WRN function via ubiquitin-dependent degradation and reveal an unexpected role of MDM2 in promoting cellular senescence through a p53-independent manner.
Assuntos
Senescência Celular/fisiologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Helicase da Síndrome de Werner/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Dano ao DNA/genética , Reparo do DNA/genética , Regulação para Baixo/genética , Etoposídeo/metabolismo , Células HCT116 , Células HEK293 , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/genética , Síndrome de Werner/metabolismoRESUMO
Reactive oxygen species generate 8-oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine), which induces G:CâT:A transversion mutations. The knockdowns of the protein responsible for Werner syndrome (WRN), a cancer-associated DNA helicase, and DNA polymerase (pol) λ, a WRN-interacting DNA pol, cause untargeted base-substitution mutations (action-at-a-distance mutations). To examine the consequences of the dual reductions of WRN and pol λ for the mutations caused by GO, siRNAs against both proteins were introduced into human U2OS cells. A replicable plasmid DNA with the oxidised nucleobase in a unique position of the supF gene was then introduced into the double knockdown cells. The amplified DNA recovered from the cells was used to transform a bacterial indicator strain. The mutant frequency and the subsequent sequence analysis revealed that the double knockdown additively promoted the G:CâT:A substitution at the GO position and increased the action-at-a-distance mutations to a level similar to that of the single WRN knockdown. Thus, WRN and DNA pol λ seem to suppress the targeted G:CâT:A mutation at least in part independently and reduce the untargeted mutations via an identical pathway.