RESUMO
The accumulation of senescent cells is a major cause of age-related inflammation and predisposes to a variety of age-related diseases1. However, little is known about the molecular basis underlying this accumulation and its potential as a target to ameliorate the ageing process. Here we show that senescent cells heterogeneously express the immune checkpoint protein programmed death-ligand 1 (PD-L1) and that PD-L1+ senescent cells accumulate with age in vivo. PD-L1- cells are sensitive to T cell surveillance, whereas PD-L1+ cells are resistant, even in the presence of senescence-associated secretory phenotypes (SASP). Single-cell analysis of p16+ cells in vivo revealed that PD-L1 expression correlated with higher levels of SASP. Consistent with this, administration of programmed cell death protein 1 (PD-1) antibody to naturally ageing mice or a mouse model with normal livers or induced nonalcoholic steatohepatitis reduces the total number of p16+ cells in vivo as well as the PD-L1+ population in an activated CD8+ T cell-dependent manner, ameliorating various ageing-related phenotypes. These results suggest that the heterogeneous expression of PD-L1 has an important role in the accumulation of senescent cells and inflammation associated with ageing, and the elimination of PD-L1+ senescent cells by immune checkpoint blockade may be a promising strategy for anti-ageing therapy.
Assuntos
Envelhecimento , Antígeno B7-H1 , Fenótipo , Receptor de Morte Celular Programada 1 , Animais , Camundongos , Envelhecimento/imunologia , Envelhecimento/metabolismo , Envelhecimento/patologia , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Inflamação/patologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/metabolismo , Análise de Célula Única , Hepatopatia Gordurosa não Alcoólica , Fígado , RejuvenescimentoRESUMO
BACKGROUND: The heart comprises many types of cells such as cardiomyocytes, endothelial cells (ECs), fibroblasts, smooth muscle cells, pericytes, and blood cells. Every cell type responds to various stressors (eg, hemodynamic overload and ischemia) and changes its properties and interrelationships among cells. To date, heart failure research has focused mainly on cardiomyocytes; however, other types of cells and their cell-to-cell interactions might also be important in the pathogenesis of heart failure. METHODS: Pressure overload was imposed on mice by transverse aortic constriction and the vascular structure of the heart was examined using a tissue transparency technique. Functional and molecular analyses including single-cell RNA sequencing were performed on the hearts of wild-type mice and EC-specific gene knockout mice. Metabolites in heart tissue were measured by capillary electrophoresis-time of flight-mass spectrometry system. The vaccine was prepared by conjugating the synthesized epitope peptides with keyhole limpet hemocyanin and administered to mice with aluminum hydroxide as an adjuvant. Tissue samples from heart failure patients were used for single-nucleus RNA sequencing to examine gene expression in ECs and perform pathway analysis in cardiomyocytes. RESULTS: Pressure overload induced the development of intricately entwined blood vessels in murine hearts, leading to the accumulation of replication stress and DNA damage in cardiac ECs. Inhibition of cell proliferation by a cyclin-dependent kinase inhibitor reduced DNA damage in ECs and ameliorated transverse aortic constriction-induced cardiac dysfunction. Single-cell RNA sequencing analysis revealed upregulation of Igfbp7 (insulin-like growth factor-binding protein 7) expression in the senescent ECs and downregulation of insulin signaling and oxidative phosphorylation in cardiomyocytes of murine and human failing hearts. Overexpression of Igfbp7 in the murine heart using AAV9 (adeno-associated virus serotype 9) exacerbated cardiac dysfunction, while EC-specific deletion of Igfbp7 and the vaccine targeting Igfbp7 ameliorated cardiac dysfunction with increased oxidative phosphorylation in cardiomyocytes under pressure overload. CONCLUSIONS: Igfbp7 produced by senescent ECs causes cardiac dysfunction and vaccine therapy targeting Igfbp7 may be useful to prevent the development of heart failure.
Assuntos
Insuficiência Cardíaca , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Camundongos Knockout , Animais , Insuficiência Cardíaca/metabolismo , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Camundongos , Humanos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Camundongos Endogâmicos C57BL , Masculino , Modelos Animais de DoençasRESUMO
The proper location and timing of Dnmt1 activation are essential for DNA methylation maintenance. We demonstrate here that Dnmt1 utilizes two-mono-ubiquitylated histone H3 as a unique ubiquitin mark for its recruitment to and activation at DNA methylation sites. The crystal structure of the replication foci targeting sequence (RFTS) of Dnmt1 in complex with H3-K18Ub/23Ub reveals striking differences to the known ubiquitin-recognition structures. The two ubiquitins are simultaneously bound to the RFTS with a combination of canonical hydrophobic and atypical hydrophilic interactions. The C-lobe of RFTS, together with the K23Ub surface, also recognizes the N-terminal tail of H3. The binding of H3-K18Ub/23Ub results in spatial rearrangement of two lobes in the RFTS, suggesting the opening of its active site. Actually, incubation of Dnmt1 with H3-K18Ub/23Ub increases its catalytic activity in vitro. Our results therefore shed light on the essential role of a unique ubiquitin-binding module in DNA methylation maintenance.
Assuntos
DNA (Citosina-5-)-Metiltransferases/química , Metilação de DNA , Histonas/química , Ubiquitina/química , Animais , Cristalografia por Raios X , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Ligação Proteica , Estrutura Quaternária de Proteína , Ubiquitina/genética , Ubiquitina/metabolismo , Xenopus laevisRESUMO
Cellular senescence plays a central role in aging and geriatric diseases. Senolysis is a promising new strategy that selectively kills and eliminates senescent cells to control aging. To date, various senolytic drugs have been discovered and shown efficacy. This review highlights how we can benefit from senolysis.
Assuntos
Envelhecimento , Senescência Celular , Humanos , IdosoRESUMO
Aging is a life phenomenon that occurs in most living organisms and is a major risk factor for many diseases, including cancer. Cellular senescence is a cellular trait induced by various genomic and epigenetic stresses. Senescent cells are characterized by irreversible cell growth arrest and excessive secretion of inflammatory cytokines (senescence-associated secretory phenotypes, SASP). Chronic tissue microinflammation induced by SASP contributes to the pathogenesis of a variety of age-related diseases, including cancer. Senolysis is a promising new strategy to selectively eliminate senescent cells in order to suppress chronic inflammation, suggesting its potential use as an anticancer therapy. This review summarizes recent findings on the molecular basis of senescence in cancer cells and senolysis.
Assuntos
Senescência Celular , Neoplasias , Fenótipo Secretor Associado à Senescência , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Animais , Inflamação/metabolismo , Envelhecimento/metabolismo , Citocinas/metabolismoRESUMO
DNA methylation is a chemical modification that defines cell type and lineage through the control of gene expression and genome stability. Disruption of DNA methylation control mechanisms causes a variety of diseases, including cancer. Cancer cells are characterized by aberrant DNA methylation (i.e., genome-wide hypomethylation and site-specific hypermethylation), mainly targeting CpG islands in gene expression regulatory elements. In particular, the early findings that a variety of tumor suppressor genes (TSGs) are targets of DNA hypermethylation in cancer led to the proposal of a model in which aberrant DNA methylation promotes cellular oncogenesis through TSGs silencing. However, recent genome-wide analyses have revealed that this classical model needs to be reconsidered. In this review, we will discuss the molecular mechanisms of DNA methylation abnormalities in cancer as well as their therapeutic potential.
Assuntos
Metilação de DNA , Neoplasias , Ilhas de CpG/genética , Metilação de DNA/genética , Estudo de Associação Genômica Ampla , Humanos , Neoplasias/genética , Neoplasias/metabolismoRESUMO
BACKGROUND: Invasive lobular carcinoma (ILC) is distinct from invasive ductal carcinoma (IDC) in terms of their hormonal microenvironments that may require different therapeutic strategies. We previously reported that selective estrogen receptor modulator (SERM) function requires F-box protein 22 (Fbxo22). Here, we investigated the role of Fbxo22 as a potential biomarker contributing to the resistance to endocrine therapy in ILC. METHODS: A total of 302 breast cancer (BC) patients including 150 ILC were recruited in the study. Fbxo22 expression and clinical information were analyzed to elucidate whether Fbxo22 negativity could be a prognostic factor or there were any correlations among clinical variables and SERM efficacy. RESULTS: Fbxo22 negativity was significantly higher in ILC compared with IDC (58.0% vs. 27.0%, P < 0.001) and higher in postmenopausal patients than premenopausal patients (64.1% vs. 48.2%, P = 0.041). In the ILC cohort, Fbxo22-negative patients had poorer overall survival (OS) than Fbxo22-positive patients, with 10-year OS rates of 77.4% vs. 93.6% (P = 0.055). All patients treated with SERMs, Fbxo22 negativity resulted in a poorer outcome, with 10-year OS rates of 81.3% vs. 92.3% (P = 0.032). In multivariate analysis regarding recurrence-free survival (RFS) in ILC patients, Fbxo22 status was independently predictive of survival as well as lymph node metastasis. CONCLUSION: Fbxo22 negativity significantly impacts on survival in BC patients with IDC and ILC, and the disadvantage was enhanced among ILC postmenopausal women or patients treated with SERMs. The findings suggest that different therapeutic strategies might be needed according to the different histopathological types when considering adjuvant endocrine therapy.
Assuntos
Neoplasias da Mama , Carcinoma Ductal de Mama , Carcinoma Lobular , Feminino , Humanos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma Lobular/patologia , Moduladores Seletivos de Receptor Estrogênico/uso terapêutico , Carcinoma Ductal de Mama/patologia , Resultado do Tratamento , Microambiente TumoralRESUMO
Ubiquitin-like with PHD and RING finger domain-containing protein 1 (UHRF1)-dependent DNA methylation is essential for maintaining cell fate during cell proliferation. Developmental pluripotency-associated 3 (DPPA3) is an intrinsically disordered protein that specifically interacts with UHRF1 and promotes passive DNA demethylation by inhibiting UHRF1 chromatin localization. However, the molecular basis of how DPPA3 interacts with and inhibits UHRF1 remains unclear. We aimed to determine the structure of the mouse UHRF1 plant homeodomain (PHD) complexed with DPPA3 using nuclear magnetic resonance. Induced α-helices in DPPA3 upon binding of UHRF1 PHD contribute to stable complex formation with multifaceted interactions, unlike canonical ligand proteins of the PHD domain. Mutations in the binding interface and unfolding of the DPPA3 helical structure inhibited binding to UHRF1 and its chromatin localization. Our results provide structural insights into the mechanism and specificity underlying the inhibition of UHRF1 by DPPA3.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Dedos de Zinco PHD , Camundongos , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Cromatina , Metilação de DNA , Proteínas Cromossômicas não Histona/metabolismoRESUMO
Cellular senescence is involved in the pathogenesis of various diseases, including acute kidney injury (AKI). AKI is defined as a sudden loss of kidney function. In severe AKI, irreversible loss of kidney cells can occur. Cellular senescence might contribute to this maladaptive tubular repair, though, its pathophysiological role in vivo is incompletely understood. In this study, we used p16-CreERT2-tdTomato mice in which cells with high p16 expression, a prototypical senescent marker, are labeled with tdTomato fluorescence. Then, we induced AKI by rhabdomyolysis and traced the cells with high p16 expression following AKI. We proved that the induction of senescence was observed predominantly in proximal tubular epithelial cells (PTECs) and occurred in a relatively acute phase within 1-3 days after AKI. These acute senescent PTECs were spontaneously eliminated by day 15. On the contrary, the generation of senescence in PTECs persisted during the chronic recovery phase. We also confirmed that the kidney function did not fully recover on day 15. These results suggest that the chronic generation of senescent PTECs might contribute to maladaptive recovery from AKI and lead to chronic kidney disease progression.
Assuntos
Injúria Renal Aguda , Insuficiência Renal Crônica , Rabdomiólise , Camundongos , Animais , Injúria Renal Aguda/patologia , Rim/patologia , Insuficiência Renal Crônica/patologia , Senescência Celular/fisiologia , Rabdomiólise/complicações , Rabdomiólise/metabolismo , Rabdomiólise/patologiaRESUMO
Cancer survivors who received chemotherapy, such as the anthracycline doxorubicin (DOX), have an increased risk of developing complications later in life, including the development of chronic metabolic diseases. Although the etiology of this increased risk for late metabolic complications in cancer survivors is poorly understood, a causal role of therapy-induced senescent cells has been suggested. To study the role of cellular senescence in chemotherapy-induced metabolic complications, young adult female low-density lipoprotein receptor-deficient (Ldlr-/-)-p16-3MR mice, in which p16Ink4a-positive (p16Ink4a+) senescent cells can be genetically eliminated, were treated with four weekly injections of DOX (2.5 mg/kg) followed by a high-fat high-cholesterol diet for 12 weeks. While DOX treatment induced known short-term effects, such as reduction in body weight, gonadal fat mass, and adipose tissue inflammation, it was not associated with significant long-term effects on glucose homeostasis, hepatic steatosis, or atherosclerosis. We further found no evidence of DOX-induced accumulation of p16Ink4a+-senescent cells at 1 or 12 weeks after DOX treatment. Neither did we observe an effect of elimination of p16Ink4a+-senescent cells on the development of diet-induced cardiometabolic complications in DOX-treated mice. Other markers for senescence were generally also not affected except for an increase in p21 and Cxcl10 in gonadal white adipose tissue long-term after DOX treatment. Together, our study does not support a significant role for p16Ink4a+-senescent cells in the development of diet-induced cardiometabolic disease in young adult DOX-treated female Ldlr-/- mice. These findings illustrate the need of further studies to understand the link between cancer therapy and cardiometabolic disease development in cancer survivors.
Assuntos
Doenças Cardiovasculares , Inibidor p16 de Quinase Dependente de Ciclina , Camundongos , Feminino , Animais , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/farmacologia , Senescência Celular , Doxorrubicina/toxicidade , Antraciclinas/farmacologiaRESUMO
DNA damage results in activation or suppression of transcription of a large number of genes. Transcriptional activation has been well characterized in the context of sequence-specific DNA-bound activators, whereas mechanisms of transcriptional suppression are largely unexplored. We show here that DNA damage rapidly reduces histone H3 Threonine 11 (T11) phosphorylation. This correlates with repression of genes, including cyclin B1 and cdk1. H3-T11 phosphorylation occurs throughout the cell cycle and is Chk1 dependent in vivo. Following DNA damage, Chk1 undergoes rapid chromatin dissociation, concomitant with reduced H3-T11 phosphorylation. Furthermore, we find that loss of H3-T11 phosphorylation correlates with reduced binding of the histone acetyltransferase GCN5 at cyclin B1 and cdk1 promoters and reduced H3-K9 acetylation. We propose a mechanism for Chk1 as a histone kinase, responsible for DNA-damage-induced transcriptional repression by loss of histone acetylation.
Assuntos
Histonas/metabolismo , Proteínas Quinases/química , Proteínas Quinases/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Transcrição Gênica , Adenoviridae/genética , Animais , Células Cultivadas , Quinase 1 do Ponto de Checagem , Meios de Cultura Livres de Soro , Dano ao DNA , Embrião de Mamíferos , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Regulação da Expressão Gênica , Células HCT116 , Histonas/genética , Humanos , Camundongos , Modelos Genéticos , Fosforilação/efeitos da radiação , Proteínas Quinases/análise , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Especificidade por Substrato , Raios UltravioletaRESUMO
DNA ligase 1 (LIG1) is known as the major DNA ligase responsible for Okazaki fragment joining. Recent studies have implicated LIG3 complexed with XRCC1 as an alternative player in Okazaki fragment joining in cases where LIG1 is not functional, although the underlying mechanisms are largely unknown. Here, using a cell-free system derived from Xenopus egg extracts, we demonstrated the essential role of PARP1-HPF1 in LIG3-dependent Okazaki fragment joining. We found that Okazaki fragments were eventually ligated even in the absence of LIG1, employing in its place LIG3-XRCC1, which was recruited onto chromatin. Concomitantly, LIG1 deficiency induces ADP-ribosylation of histone H3 in a PARP1-HPF1-dependent manner. The depletion of PARP1 or HPF1 resulted in a failure to recruit LIG3 onto chromatin and a subsequent failure in Okazaki fragment joining in LIG1-depleted extracts. Importantly, Okazaki fragments were not ligated at all when LIG1 and XRCC1 were co-depleted. Our results suggest that a unique form of ADP-ribosylation signaling promotes the recruitment of LIG3 on chromatin and its mediation of Okazaki fragment joining as a backup system for LIG1 perturbation.
Assuntos
DNA Ligase Dependente de ATP/metabolismo , DNA/metabolismo , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Sistema Livre de Células , Poli(ADP-Ribose) Polimerase-1/metabolismo , Xenopus laevisRESUMO
DNA lesions impact on local transcription and the damage-induced transcriptional repression facilitates efficient DNA repair. However, how chromatin dynamics cooperates with these two events remained largely unknown. We here show that histone H2A acetylation at K118 is enriched in transcriptionally active regions. Under DNA damage, the RSF1 chromatin remodeling factor recruits HDAC1 to DSB sites. The RSF1-HDAC1 complex induces the deacetylation of H2A(X)-K118 and its deacetylation is indispensable for the ubiquitination of histone H2A at K119. Accordingly, the acetylation mimetic H2A-K118Q suppressed the H2A-K119ub level, perturbing the transcriptional repression at DNA lesions. Intriguingly, deacetylation of H2AX at K118 also licenses the propagation of γH2AX and recruitment of MDC1. Consequently, the H2AX-K118Q limits DNA repair. Together, the RSF1-HDAC1 complex controls the traffic of the DNA damage response and transcription simultaneously in transcriptionally active chromatins. The interplay between chromatin remodelers and histone modifiers highlights the importance of chromatin versatility in the maintenance of genome integrity.
Assuntos
Montagem e Desmontagem da Cromatina , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Epigênese Genética , Regulação da Expressão Gênica , Proteínas Nucleares/genética , Transativadores/genética , Acetilação , Animais , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Células HEK293 , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Lisina/genética , Lisina/metabolismo , Camundongos , Microscopia Confocal , Células NIH 3T3 , Proteínas Nucleares/metabolismo , Ligação Proteica , Interferência de RNA , Transativadores/metabolismo , UbiquitinaçãoRESUMO
Senescence is a state of permanent growth arrest and is a pivotal part of the antitumorigenic barrier in vivo. Although the tumor suppressor activities of p53 and pRb family proteins are essential for the induction of senescence, molecular mechanisms by which these proteins induce senescence are still not clear. Using time-lapse live-cell imaging, we demonstrate here that normal human diploid fibroblasts (HDFs) exposed to various senescence-inducing stimuli undergo a mitosis skip before entry into permanent cell-cycle arrest. This mitosis skip is mediated by both p53-dependent premature activation of APC/C(Cdh1) and pRb family protein-dependent transcriptional suppression of mitotic regulators. Importantly, mitotic skipping is necessary and sufficient for senescence induction. p16 is only required for maintenance of senescence. Analysis of human nevi also suggested the role of mitosis skip in in vivo senescence. Our findings provide decisive evidence for the molecular basis underlying the induction and maintenance of cellular senescence.
Assuntos
Senescência Celular , Mitose/fisiologia , Pontos de Checagem do Ciclo Celular , Inibidor p16 de Quinase Dependente de Ciclina , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiologia , Imagem com Lapso de Tempo , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/fisiologiaRESUMO
DNA damage induces transcriptional repression of E2F1 target genes and a reduction in histone H3-Thr11 phosphorylation (H3-pThr11 ) at E2F1 target gene promoters. Dephosphorylation of H3-pThr11 is partly mediated by Chk1 kinase and protein phosphatase 1γ (PP1γ) phosphatase. Here, we isolated NIPP1 as a regulator of PP1γ-mediated H3-pThr11 by surveying nearly 200 PP1 interactor proteins. We found that NIPP1 inhibits PP1γ-mediated dephosphorylation of H3-pThr11 both in vivo and in vitro. By generating NIPP1-depleted cells, we showed that NIPP1 is required for cell proliferation and the expression of E2F1 target genes. Upon DNA damage, activated protein kinase A (PKA) phosphorylated the NIPP1-Ser199 residue, adjacent to the PP1 binding motif (RVxF), and triggered the dissociation of NIPP1 from PP1γ, leading to the activation of PP1γ. Furthermore, the inhibition of PKA activity led to the activation of E2F target genes. Statistical analysis confirmed that the expression of NIPP1 was positively correlated with E2F target genes. Taken together, these findings demonstrate that the PP1 regulatory subunit NIPP1 modulates E2F1 target genes by linking PKA and PP1γ during DNA damage.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dano ao DNA , Fator de Transcrição E2F1/genética , Endorribonucleases/metabolismo , Histonas/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteína Fosfatase 1/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sistemas CRISPR-Cas , Proliferação de Células , Células Cultivadas , Quinase 1 do Ponto de Checagem/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Endorribonucleases/deficiência , Endorribonucleases/isolamento & purificação , Repressão Epigenética , Regulação da Expressão Gênica , Humanos , Fosfoproteínas Fosfatases/deficiência , Fosfoproteínas Fosfatases/isolamento & purificação , Fosforilação , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/isolamento & purificação , Receptores de Neuropeptídeo Y/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Raios UltravioletaRESUMO
DNA methylation controls gene expression, and once established, DNA methylation patterns are faithfully copied during DNA replication by the maintenance DNA methyltransferase Dnmt1. In vivo, Dnmt1 interacts with Uhrf1, which recognizes hemimethylated CpGs. Recently, we reported that Uhrf1-catalyzed K18- and K23-ubiquitinated histone H3 binds to the N-terminal region (the replication focus targeting sequence, RFTS) of Dnmt1 to stimulate its methyltransferase activity. However, it is not yet fully understood how ubiquitinated histone H3 stimulates Dnmt1 activity. Here, we show that monoubiquitinated histone H3 stimulates Dnmt1 activity toward DNA with multiple hemimethylated CpGs but not toward DNA with only a single hemimethylated CpG, suggesting an influence of ubiquitination on the processivity of Dnmt1. The Dnmt1 activity stimulated by monoubiquitinated histone H3 was additively enhanced by the Uhrf1 SRA domain, which also binds to RFTS. Thus, Dnmt1 activity is regulated by catalysis (ubiquitination)-dependent and -independent functions of Uhrf1.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Histonas/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Replicação do DNA , Histonas/fisiologia , Humanos , Ligação Proteica , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
The accumulation of epigenetic alterations is one of the major causes of tumorigenesis. Aberrant DNA methylation patterns cause genome instability and silencing of tumor suppressor genes in various types of tumors. Therefore, drugs that target DNA methylation-regulating factors have great potential for cancer therapy. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) is an essential factor for DNA methylation maintenance. UHRF1 is overexpressed in various cancer cells and down-regulation of UHRF1 in these cells reactivates the expression of tumor suppressor genes, thus UHRF1 is a promising target for cancer therapy. We have previously shown that interaction between the tandem Tudor domain (TTD) of UHRF1 and DNA ligase 1 (LIG1) di/trimethylated on Lys126 plays a key role in the recruitment of UHRF1 to replication sites and replication-coupled DNA methylation maintenance. An arginine binding cavity (Arg-binding cavity) of the TTD is essential for LIG1 interaction, thus the development of inhibitors that target the Arg-binding cavity could potentially repress UHRF1 function in cancer cells. To develop such an inhibitor, we performed in silico screening using not only static but also dynamic metrics based on all-atom molecular dynamics simulations, resulting in efficient identification of 5-amino-2,4-dimethylpyridine (5A-DMP) as a novel TTD-binding compound. Crystal structure of the TTD in complex with 5A-DMP revealed that the compound stably bound to the Arg-binding cavity of the TTD. Furthermore, 5A-DMP inhibits the full-length UHRF1:LIG1 interaction in Xenopus egg extracts. Our study uncovers a UHRF1 inhibitor which can be the basis of future experiments for cancer therapy.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/antagonistas & inibidores , DNA Ligase Dependente de ATP/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Simulação de Dinâmica Molecular , Piridinas/farmacologia , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , DNA Ligase Dependente de ATP/metabolismo , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Piridinas/química , Relação Estrutura-Atividade , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , XenopusRESUMO
Ubiquitin-dependent protein degradation has been implicated in the control of various cellular processes such as cell cycle control, transcriptional regulation, DNA damage repair, and apoptosis, many of which are involved in the initiation, progression, metastasis, and drug resistance of cancers. E3 ubiquitin ligases are known to be the second most prevalent cancer-related functional gene family next to protein kinases. Of these, FBXO22, an F-box receptor subunit of SCF E3 ligase, has recently been proposed to play a critical role in multiple aspects related to cancer development and therapy response. Firstly, FBXO22 is a key regulator of senescence induction through ubiquitylation of p53 for degradation. FBXO22 also acts as a molecular switch for the antagonistic and agonistic actions of selective estrogen receptor modulators (SERM) and determines the sensitivity of breast cancer to SERM by ubiquitylating KDM4B complexed with unliganded or SERMs-bound estrogen receptor (ER). Furthermore, FBXO22 binds to Bach1, a pro-metastatic transcription factor, suppressing Bach1-driven metastasis of lung adenocarcinoma, and loss of FBXO22 facilitates metastasis. These findings, as well as other reports, unveiled strikingly important roles of FBXO22 in cancer development and therapeutic strategy. In this review, we summarize recent findings of how FBXO22 regulates major cancer suppression pathways.
Assuntos
Epigênese Genética , Proteínas F-Box/metabolismo , Neoplasias/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/patologia , Movimento Celular/genética , Senescência Celular/genética , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Neoplasias/patologia , Subunidades Proteicas/metabolismo , Proteólise , Receptores de Estrogênio/metabolismo , Moduladores Seletivos de Receptor Estrogênico/metabolismo , Transdução de Sinais/genética , UbiquitinaçãoRESUMO
SAD kinases regulate presynaptic vesicle clustering and neuronal polarization. A previous report demonstrated that Sada-/- and Sadb-/- double-mutant mice showed perinatal lethality with a severe defect in axon/dendrite differentiation, but their single mutants did not. These results indicated that they were functionally redundant. Surprisingly, we show that on a C57BL/6N background, SAD-A is essential for cortical development whereas SAD-B is dispensable. Sada-/- mice died within a few days after birth. Their cortical lamination pattern was disorganized and radial migration of cortical neurons was perturbed. Birth date analyses with BrdU and in utero electroporation using pCAG-EGFP vector showed a delayed migration of cortical neurons to the pial surface in Sada-/- mice. Time-lapse imaging of these mice confirmed slow migration velocity in the cortical plate. While the neurites of hippocampal neurons in Sada-/- mice could ultimately differentiate in culture to form axons and dendrites, the average length of their axons was shorter than that of the wild type. Thus, analysis on a different genetic background than that used initially revealed a nonredundant role for SAD-A in neuronal migration and differentiation.
Assuntos
Movimento Celular/fisiologia , Córtex Cerebral/embriologia , Córtex Cerebral/enzimologia , Neurônios/enzimologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Axônios/enzimologia , Células Cultivadas , Feminino , Isoenzimas , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genéticaRESUMO
SIGNIFICANCE: Our study revealed that the validity of a new Japanese visual-perceptual test was acceptable. Visual-perceptual abilities are important to activities of daily living; thus, accurate assessment of visual perception is especially important for children with neurodevelopmental disorders and particularly so for those with learning problems. PURPOSE: Visual perception refers to the process by which one receives visual information through sensory impulses and then translates those impulses into meaning based on a previously developed view of the environment. A problem in Japan is the paucity of visual perception tests that use normative data from children who are native Japanese. The Wide-range Assessment of Vision-related Essential Skills (WAVES), which measures visual perception and eye-hand coordination skills and is based on Japanese normative data, was recently published in Japan. The validity of this test has not been comprehensively established. METHODS: To investigate the validity of the WAVES, we used the Pearson correlation coefficient to calculate the degrees of association among WAVES scaled and index scores compared with Developmental Test of Visual Perception, Third Edition, scores from 108 elementary school children with symptoms of learning problems. Participants were recruited at Osaka Medical College Learning Disability Center and Sakai Seikeikai Learning Disability Center. RESULTS: The concurrent validity of the WAVES was supported by moderate correlation (r = 0.67, P < .01) between the total scores for visual perception and eye-hand coordination index from the WAVES and general visual perception index from the Developmental Test of Visual Perception, Third Edition, even though a correlation analysis of subtests found differences between the two tests. CONCLUSIONS: Our results showed that the indices from the two tests measured nearly the same underlying visual-perceptual constructs and indicated that the WAVES had acceptable levels of concurrent validity.