RESUMO
DNA replication is a major contributor to genomic instability, and protection against DNA replication perturbation is essential for normal cell division. Certain types of replication stress agents, such as aphidicolin and hydroxyurea, have been shown to cause reversible replication fork stalling, wherein replisome complexes are stably maintained with competence to restart in the S phase of the cell cycle. If these stalled forks persist into the M phase without a replication restart, replisomes are disassembled in a p97-dependent pathway and under-replicated DNA is subjected to mitotic DNA repair synthesis. Here, using Xenopus egg extracts, we investigated the consequences that arise when stalled forks are released simultaneously with the induction of mitosis. Ara-cytidine-5'-triphosphate-induced stalled forks were able to restart with the addition of excess dCTP during early mitosis before the nuclear envelope breakdown (NEB). However, stalled forks could no longer restart efficiently after the NEB. Although replisome complexes were finally disassembled in a p97-dependent manner during mitotic progression whether or not fork stalling was relieved, the timing of the NEB was delayed with the ongoing forks, rather than the stalled forks, and the delay was dependent on Wee1/Myt1 kinase activities. Thus, ongoing DNA replication was found to be directly linked to the regulation of Wee1/Myt1 kinases to modulate cyclin-dependent kinase activities because of which DNA replication and mitosis occur in a mutually exclusive and sequential manner.
Assuntos
Replicação do DNA , Mitose , Membrana Nuclear/metabolismo , Animais , Sistema Livre de Células , Xenopus laevisRESUMO
Tissue stem cell senescence leads to stem cell exhaustion, which results in tissue homeostasis imbalance and a decline in regeneration capacity. However, whether neural stem cell (NSC) senescence occurs and causes neurogenesis reduction during aging is unknown. In this study, mice at different ages were used to detect age-related hippocampal NSC (H-NSC) senescence, as well as the function and mechanism of embryonic stem cell-derived small extracellular vesicles (ESC-sEVs) in rejuvenating H-NSC senescence. We found a progressive cognitive impairment, as well as age-related H-NSC senescence, in mice. ESC-sEV treatment significantly alleviated H-NSC senescence, recovered compromised self-renewal and neurogenesis capacities, and reversed cognitive impairment. Transcriptome analysis revealed that myelin transcription factor 1 (MYT1) is downregulated in senescent H-NSCs but upregulated by ESC-sEV treatment. In addition, knockdown of MYT1 in young H-NSCs accelerated age-related phenotypes and impaired proliferation and differentiation capacities. Mechanistically, ESC-sEVs rejuvenated senescent H-NSCs partly by transferring SMAD family members 4 (SMAD4) and 5 (SMAD5) to activate MYT1, which downregulated egl-9 family hypoxia inducible factor 3 (Egln3), followed by activation of hypoxia inducible factor 2 subunit α (HIF-2α), nicotinamide phosphoribosyl transferase (NAMPT), and sirtuin 1 (Sirt1) successively. Taken together, our results indicated that H-NSC senescence caused cellular exhaustion, neurogenesis reduction, and cognitive impairment during aging, which can be reversed by ESC-sEVs. Thus, ESC-sEVs may be promising therapeutic candidates for age-related diseases.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Vesículas Extracelulares/metabolismo , Hipocampo/citologia , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Células-Tronco Neurais/metabolismo , Sirtuína 1/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/genética , Senescência Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Transdução de SinaisRESUMO
Gliomas portray a large and heterogeneous group of CNS tumors, encompassing a wide range of low- to high-grade tumors, as defined by histological and molecular characteristics. The identification of signature mutations and other molecular abnormalities has largely impacted tumor classification, diagnosis, and therapy. Transcription factors (TFs) are master regulators of gene expression programs, which ultimately shape cell fate and homeostasis. A variety of TFs have been detected to be aberrantly expressed in brain tumors, being highly implicated in critical pathological aspects and progression of gliomas. Herein, we describe a selection of oncogenic (GLI-1/2/3, E2F1-8, STAT3, and HIF-1/2) and tumor suppressor (NFI-A/B, TBXT, MYT1, and MYT1L) TFs that are deregulated in gliomas and are subsequently associated with tumor development, progression, and migratory potential. We further discuss the current targeting options against these TFs, including chemical (Bortezomib) and natural (Plumbagin) compounds, small molecules, and inhibitors, and address their potential implications in glioma therapy.
Assuntos
Neoplasias Encefálicas , Glioma , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Genes Supressores de Tumor , Glioma/tratamento farmacológico , Glioma/genética , Glioma/metabolismo , Humanos , Mutação , OncogenesRESUMO
Major depressive disorder (MDD) and bipolar disorder (BPD) are among the most debilitating mental conditions. Diagnostic criteria for MDD include psychological and physical symptoms, such as low mood and changes in appetite or sleep, respectively. BPD in addition to periods of depression represents episodes of mania or hypomania, and elevation in mood and energy levels are associated with this condition. Dysregulation in adult neurogenesis and myelination have been reported in psychiatric disorders. As a key factor in neurogenesis, it was hypothesized that Myt1 gene expression may be altered in these conditions. Using Real-time PCR, Myt1 expression level in 100 MDD patients and 100 BPD patients, compared with healthy control (HC) individuals was evaluated. Results demonstrate significant downregulation of Myt1 in MDD and BPD. Logistic regression analysis and binary classification evaluation reveal potential risk factor and biomarker characteristics of Myt1, respectively. Moreover, forward and backward digit span results denote a significant reduction in the function of working memory (WM) of MDD and BPD subjects. Correlation analysis revealed a significant association between Myt1 downregulation and WM disruption in the affected individuals. In conclusion, due to its altered role in neurogenesis, downregulation of Myt1 can be associated with the pathology of MDD and BPD.
Assuntos
Transtorno Bipolar , Proteínas de Ligação a DNA , Transtorno Depressivo Maior , Fatores de Transcrição , Adulto , Biomarcadores , Transtorno Bipolar/diagnóstico , Transtorno Bipolar/genética , Transtorno Bipolar/psicologia , Proteínas de Ligação a DNA/genética , Transtorno Depressivo Maior/diagnóstico , Transtorno Depressivo Maior/genética , Transtorno Depressivo Maior/psicologia , Humanos , Fatores de Risco , Fatores de Transcrição/genéticaRESUMO
Oculo-auriculo-vertebral spectrum (OAVS) is a developmental disorder characterized by anomalies mainly involving the structures derived from the first and second pharyngeal arches. The spectrum presents with heterogeneous clinical features and complex etiology with genetic factors not yet completely understood. To date, MYT1 is the most important gene unambiguously associated with the spectrum and with functional data confirmation. In this work, we aimed to identify new single nucleotide variants (SNVs) affecting MYT1 in a cohort of 73 Brazilian patients diagnosed with OAVS. In addition, we investigated copy number variations (CNVs) encompassing this gene or its cis-regulatory elements and compared the frequency of these events in patients versus a cohort of 455 Brazilian control individuals. A new SNV, predicted as likely deleterious, was identified in five unrelated patients with OAVS. All five patients presented hearing impairment and orbital asymmetry suggesting an association with the variant. CNVs near MYT1, located in its neighboring topologically associating domain (TAD), were found to be enriched in patients when compared to controls, indicating a possible involvement of this region with OAVS pathogenicity. Our findings highlight the genetic complexity of the spectrum that seems to involve more than one variant type and inheritance patterns.
Assuntos
Proteínas de Ligação a DNA/genética , Predisposição Genética para Doença , Síndrome de Goldenhar/genética , Fatores de Transcrição/genética , Região Branquial/patologia , Brasil/epidemiologia , Variações do Número de Cópias de DNA/genética , Feminino , Síndrome de Goldenhar/epidemiologia , Síndrome de Goldenhar/patologia , Humanos , Masculino , Fenótipo , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
Both point mutations and deletions of the MYT1L gene as well as microdeletions of chromosome band 2p25.3 including MYT1L are associated with intellectual disability, obesity, and behavioral problems. Thus, MYT1L is assumed to be the-at least mainly-causative gene in the 2p25.3 deletion syndrome. Here, we present comprehensive descriptions of nine novel individuals bearing MYT1L mutations; most of them single nucleotide variants (SNVs). This increases the number of known individuals with causative deletions or SNVs of MYT1L to 51. Since eight of the nine novel patients bear mutations affecting MYT1L only, the total number of such individuals now nearly equals the number of individuals with larger microdeletions affecting additional genes, allowing for a comprehensive phenotypic comparison of these two patient groups. For example, 55% of the individuals with mutations affecting MYT1L only were overweight or obese as compared to 86% of the individuals with larger microdeletions. A similar trend was observed regarding short stature with 5 versus 35%, respectively. However, these differences were nominally significant only after correction for multiple testing, further supporting the hypothesis that MYT1L haploinsufficiency is central to the 2p25.3 deletion phenotype. Most importantly, the large number of individuals with MYT1L mutations presented and reviewed here allowed for the delineation of a more comprehensive clinical picture. Seizures, postnatal short stature, macrocephaly, and microcephaly could be shown to be over-represented among individuals with MYT1L mutations.
Assuntos
Predisposição Genética para Doença , Deficiência Intelectual/genética , Proteínas do Tecido Nervoso/genética , Obesidade/genética , Fatores de Transcrição/genética , Adolescente , Adulto , Criança , Pré-Escolar , Deleção Cromossômica , Cromossomos Humanos Par 2/genética , Feminino , Haploinsuficiência/genética , Humanos , Deficiência Intelectual/fisiopatologia , Masculino , Análise em Microsséries , Microcefalia/genética , Microcefalia/fisiopatologia , Obesidade/fisiopatologia , Fenótipo , Mutação Puntual , Polimorfismo de Nucleotídeo Único/genética , Sequenciamento do Exoma , Adulto JovemRESUMO
Variations in MYT1L, a gene encoding a transcription factor expressed in the brain, have been associated with autism, intellectual disability, and schizophrenia. Here we provide an updated review of published reports of neuropsychiatric correlates of loss of function and duplication of MYT1L. Of 27 duplications all were partial; 33% were associated exclusively with schizophrenia, and the chromosomal locations of schizophrenia-associated duplications exhibited a distinct difference in pattern-of-location from those associated with autism and/or intellectual disability. Of 51 published heterozygous loss of function variants, all but one were associated with intellectual disability, autism, or both, and one resulted in no neuropsychiatric diagnosis. There were no reports of schizophrenia associated with loss of function variants of MYT1L (Fisher's exact p < .00001, for contrast with all reported duplications). Although the precise function of the various mutations remains unspecified, these data collectively establish the candidacy of MYT1L as a reciprocal mutation, in which schizophrenia may be engendered by partial duplications, typically involving the 3' end of the gene, while developmental disability-notably autism-is associated with both loss of function and partial duplication. Future research on the specific effects of contrasting mutations in MYT1L may provide insight into the causal origins of autism and schizophrenia.
Assuntos
Transtorno Autístico/genética , Variação Genética , Proteínas do Tecido Nervoso/genética , Esquizofrenia/genética , Fatores de Transcrição/genética , Deleção de Genes , Duplicação Gênica , Regulação da Expressão Gênica , Estudos de Associação Genética , Humanos , Deficiência Intelectual/genética , Mutação , FenótipoRESUMO
During cleavage of Xenopus laevis, the first mitotic cell cycle immediately following fertilization is approximately 90â¯min and consists of S, G2, and M phases. In contrast, the subsequent eleven cell cycles are approximately 30â¯min and consist mostly of S and M phases. The balance between Cdc25 and Wee1A/Myt1 is thought to be crucial for Xenopus first cell cycle progression; however, the role of Myt1 in this period has not been fully investigated. In this study, we examined the roles of Myt1, Wee1A, and Cdc25A in the first cell cycle of Xenopus laevis. Inhibition of Cdc25A with antisense morpholino oligonucleotides lengthened the duration of the first cell cycle to some extent, whereas it was slightly shortened by ectopic Cdc25A expression, suggesting that the low concentration of Cdc25A during the first cell cycle does not fully account for the long duration of this cycle. Using the Wee1A antisense morpholino oligonucleotide and neutralizing antibody against Myt1, we found that Myt1 phosphorylates and inhibits Cdk1 much more effectively than Wee1A during the first cell cycle in Xenopus. Taken together, these results suggest that the activity of Myt1 is predominantly responsible for the duration of the long G2 phase in the first mitotic cell cycle in Xenopus.
Assuntos
Ciclo Celular/genética , Fase G2/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Feminino , Regulação da Expressão Gênica , Mitose/genética , Oligonucleotídeos Antissenso/genética , Oócitos/citologia , Oócitos/metabolismo , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismoRESUMO
In aquaculture practices, prawn cultivation holds the major share and Penaeus monodon is the main species cultured. The decline in production of P. monodon is mainly due to the limited availability of domesticated broodstock, which is attributed to its reproductive cycle, controlled by complex coordinated signaling mechanisms. Unilateral eyestalk ablation of domesticated females held in captivity is done to induce ovary development, which has certain disadvantages, including a high rate of mortality. Thus, developing alternative techniques for eyestalk ablation in captive broodstock is necessary to induce maturation of ovary. This study exemplifies the role of 5HT along with a cocktail of inhibitors (U0126, Rp-cAMP, and LY294002) in inducing ovarian maturation. In this study, inhibition of pERK by U0126 inhibited vitellogenesis-inhibiting hormone (VIH), which in turn led to the overexpression of vitellogenin. 5HT induces steroidogenesis (estradiol-17ß) through induction of the gonadotropin-releasing hormone by activating calcium-calmodulin signaling. Steroidogenesis is also aided by synthesis of StAR protein. Estradiol-17ß stimulates the formation of the maturation-promoting factor (MPF) complex by cdc25 activation and Myt1 inactivation. LY294002 aids in keeping cdc25 activated by inhibiting calcium-calmodulin induced phosphorylation of Akt which is a negative regulator of mitogen-activated protein kinases. VIH induced activation of Myt1, through protein kinase A (PKA), was inhibited by Rp-cAMP which inhibits adenylate cyclase, thus stabilizing the activated MPF complex. To conclude, the coordinated effect of inhibitors and 5HT accelerates the development of ovary from previtellogenic to matured oocytes, yielding high quality and quantity larvae compared with eyestalk-ablated P. monodon.
Assuntos
Ovário , Serotonina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Inibidores Enzimáticos/farmacologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Ovário/efeitos dos fármacos , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Penaeidae , Fosforilação/efeitos dos fármacos , Vitelogeninas/metabolismoRESUMO
Myt1 and Myt1l (Myelin transcription factor 1, and Myt1-like) are members of a small family of closely related zinc finger transcription factors, characterized by two clusters of C2HC zinc fingers. Both are widely expressed during early embryogenesis, but are largely restricted to expression within the brain in the adult. Myt1l, as part of a three transcription factor mix, can reprogram fibroblasts to neurons and plays a role in maintaining neuronal identity. Previous analyses have indicated roles in both transcriptional activation and repression and suggested that Myt1 and Myt1l may have opposing functions in gene expression. We show that when targeted to DNA via multiple copies of the consensus Myt1/Myt1l binding site Myt1 represses transcription, whereas Myt1l activates. By targeting via a heterologous DNA binding domain we mapped an activation function in Myt1l to an amino-terminal region that is poorly conserved in Myt1. However, genome wide analyses of the effects of Myt1 and Myt1l expression in a glioblastoma cell line suggest that the two proteins have largely similar effects on endogenous gene expression. Transcriptional repression is likely mediated by binding to DNA via the known consensus site, whereas this site is not associated with the transcriptional start sites of genes with higher expression in the presence of Myt1 or Myt1l. This work suggests that these two proteins function similarly, despite differences observed in analyses based on synthetic reporter constructs.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Elementos de Resposta , Fatores de Transcrição/metabolismo , Transcrição Gênica , Células A549 , Proteínas de Ligação a DNA/genética , Glioblastoma/genética , Glioblastoma/patologia , Células HEK293 , Células HeLa , Humanos , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição/genéticaRESUMO
The genetic background of severe early-onset obesity is still incompletely understood. Deletions at 2p25.3 associate with early-onset obesity and variable intellectual disability. Myelin-transcriptor-factor-1-like (MYT1L) gene in this locus has been proposed a candidate gene for obesity. We report on a 13-year-old boy presenting with overweight already at 1 year of age (body mass index [BMI] Z-score +2.3) and obesity at 2 years of age (BMI Z-score +3.8). The patient had hyperphagia and delayed neurological, cognitive and motor development. He also had speech delay, strabismus, hyperactivity and intellectual disability. Brain MRI was normal. The parents and sister had normal BMI. Whole-genome sequencing identified in the index patient a novel de novo frameshift deletion that introduces a premature termination of translation NM_015025.2(MYT1L): c.2215_2224delACGCGCTGCC, p.(Thr739Alafs*7) in MYT1L. The frameshift variant was confirmed by Sanger sequencing. Our finding supports the association of MYT1L mutations with early-onset syndromic obesity. The identification of novel monogenic forms of childhood-onset obesity will provide insights to the involved genetic and biologic pathways.
Assuntos
Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Mutação , Proteínas do Tecido Nervoso/genética , Obesidade/diagnóstico , Obesidade/genética , Fenótipo , Fatores de Transcrição/genética , Adolescente , Idade de Início , Alelos , Índice de Massa Corporal , Criança , Pré-Escolar , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA , Estudos de Associação Genética , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Índice de Gravidade de DoençaRESUMO
BACKGROUND: Oculo-auriculo-vertebral spectrum (OAVS) is a developmental disorder involving first and second branchial arches derivatives, mainly characterised by asymmetric ear anomalies, hemifacial microsomia, ocular defects and vertebral malformations. Although numerous chromosomal abnormalities have been associated with OAVS, no causative gene has been identified so far. OBJECTIVES: We aimed to identify the first causative gene for OAVS. METHODS: As sporadic cases are mostly described in Goldenhar syndrome, we have performed whole exome sequencing (WES) on selected affected individuals and their unaffected parents, looking for de novo mutations. Candidate gene was tested through transient knockdown experiment in zebrafish using a morpholino-based approach. A functional test was developed in cell culture in order to assess deleterious consequences of mutations. RESULTS: By WES, we identified a heterozygous nonsense mutation in one patient in the myelin transcription factor 1 (MYT1) gene. Further, we detected one heterozygous missense mutation in another patient among a cohort of 169 patients with OAVS. This gene encodes the MYT1. Functional studies by transient knockdown of myt1a, homologue of MYT1 in zebrafish, led to specific craniofacial cartilage alterations. Treatment with all-trans retinoic acid (RA), a known teratogenic agent causing OAVS, led to an upregulation of cellular endogenous MYT1 expression. Additionally, cellular wild-type MYT1 overexpression induced a downregulation of RA receptor ß (RARB), whereas mutated MYT1 did not. CONCLUSION: We report MYT1 as the first gene implicated in OAVS, within the RA signalling pathway.
RESUMO
Regulation of spatiotemporal gene expression in higher eukaryotic cells is critical for the precise and orderly development of undifferentiated progenitors into committed cell types of the adult. It is well known that dynamic epigenomic regulation (including chromatin remodeling and histone modifications by transcriptional coregulator complexes) is involved in transcriptional regulation. Precisely how these coregulator complexes exert their cell type and developing stage-specific activity is largely unknown. In this study we aimed to isolate the histone demethylase lysine-specific demethylase 1 (LSD1) complex from neural cells by biochemical purification. In so doing, we identified myelin transcription factor 1 (MyT1) as a novel LSD1 complex component. MyT1 is a neural cell-specific zinc finger factor, and it forms a stable multiprotein complex with LSD1 through direct interaction. Target gene analysis using microarray and ChIP assays revealed that the Pten gene was directly regulated by the LSD1-MyT1 complex. Knockdown of either LSD1 or MyT1 derepressed the expression of endogenous target genes and inhibited cell proliferation of a neuroblastoma cell line, Neuro2a. We propose that formation of tissue-specific combinations of coregulator complexes is a critical mechanism for tissue-specific transcriptional regulation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Neurônios/enzimologia , Oxirredutases N-Desmetilantes/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Histona Desmetilases , Histonas/metabolismo , Camundongos , Neurônios/metabolismo , Oxirredutases N-Desmetilantes/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Especificidade da Espécie , Fatores de Transcrição/genéticaRESUMO
Parkin is a known tumor suppressor. However, the mechanism by which parkin acts as a tumor suppressor remains to be fully elucidated. Previously, we reported that parkin expression induces caspase-dependent apoptotic cell death in TNF-α-treated HeLa cells. However, at that time, we did not consider the involvement of parkin in cell cycle control. In the current study, we investigated whether parkin is involved in cell cycle regulation and suppression of cancer cell growth. In our cell cycle analyses, parkin expression induced G2/M cell cycle arrest in TNF-α-treated HeLa cells. To elucidate the mechanism(s) by which parkin induces this G2/M arrest, we analyzed cell cycle regulatory molecules involved in the G2/M transition. Parkin expression induced CDC2 phosphorylation which is known to inhibit CDC2 activity and cause G2/M arrest. Cyclin B1, which is degraded during the mitotic transition, accumulated in response to parkin expression, thereby indicating parkin-induced G2/M arrest. Next, we established that Myt1, which is known to phosphorylate and inhibit CDC2, increased following parkin expression. In addition, we found that parkin also induces increased Myt1 expression, G2/M arrest, and reduced cell viability in TNF-α-treated HCT15 cells. Furthermore, knockdown of parkin expression by parkin-specific siRNA decreased Myt1 expression and phosphorylation of CDC2 and resulted in recovered cell viability. These results suggest that parkin acts as a crucial molecule causing cell cycle arrest in G2/M, thereby suppressing tumor cell growth.
Assuntos
Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina-Proteína Ligases/genética , Proteína Quinase CDC2 , Sobrevivência Celular/efeitos dos fármacos , Ciclina B1/genética , Ciclina B1/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Fosforilação/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Myt1 kinase is a member of the Wee-kinase family involved in G2/M checkpoint regulation of the cell cycle. So far, no peptide substrate suitable for activity-based screening has been reported, hampering systematic development of Myt1 kinase inhibitors. Myt1 inhibitors had to be identified by using either binding assays or activity assays with expensive proteinous substrates. Here, a peptide microarray approach was used to identify peptidic Myt1 substrates. Wee1 kinase was profiled for comparison using the same technology. Myt1 hits from peptide microarray experiments were verified in solution by a fluorescence polarization assay and several peptide substrates derived from cellular proteins were identified. Subsequently, phosphorylation site determination was carried out by MS fragmentation studies and identified substrates were validated by kinase inhibitor profiling.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Peptídeos/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Proteínas de Ligação a DNA/química , Polarização de Fluorescência , Corantes Fluorescentes/química , Humanos , Peptídeos/síntese química , Peptídeos/química , Fosforilação , Análise Serial de Proteínas , Especificidade por Substrato , Fatores de Transcrição/químicaRESUMO
We previously isolated and reported a second species of the Saccharophagus genus, Saccharophagus sp. strain Myt-1. In the present study, a cellulase gene (celMytB) from the genomic DNA of Myt-1 was cloned and characterized. The DNA sequence fragment contained an open reading frame of 1,893 bp that encoded a protein of 631 amino acids with an estimated molecular mass of 66.8 kDa. The deduced protein, CelMytB, had a catalytic domain that contained a conserved signature sequence (VIYEIYNEPL) of glycosyl hydrolase family 5 and a CBM6 cellulose binding module. CelMytB showed optimal activity at 55 °C and pH 6.5, which is similar to the optimal temperature and pH profile of cel5H, an endoglucanase from the closely related S. degradans 2-40. However, the cellulase (degradation of soluble cellulose) and avicelase (degradation of crystalline cellulose) activities of CelMytB were about 3-fold and 100-fold higher, respectively, than the equivalent activities of cel5H. Moreover, CelMytB could degrade xylan. From the zymogram results, we speculated that the catalytic domain of CelMytB had high activity even without the cellulose binding module. The presence of some detergents stimulated the cellulase activity of CelMytB.
RESUMO
BACKGROUND: The cell cycle is tightly regulated by checkpoints, which play a vital role in controlling its progression and timing. Cancer cells exploit the G2/M checkpoint, which serves as a resistance mechanism against genotoxic anticancer treatments, allowing for DNA repair prior to cell division. Manipulating cell cycle timing has emerged as a potential strategy to augment the effectiveness of DNA damage-based therapies. METHODS: In this study, we conducted a forward genome-wide CRISPR/Cas9 screening with repeated exposure to the alkylating agent temozolomide (TMZ) to investigate the mechanisms underlying tumor cell survival under genotoxic stress. RESULTS: Our findings revealed that canonical DNA repair pathways, including the Ataxia-telangiectasia mutated (ATM)/Fanconi and mismatch repair, determine cell fate under genotoxic stress. Notably, we identified the critical role of PKMYT1, in ensuring cell survival. Depletion of PKMYT1 led to overwhelming TMZ-induced cytotoxicity in cancer cells. Isobologram analysis demonstrated potent drug synergy between alkylating agents and a Myt1 kinase inhibitor, RP-6306. Mechanistically, inhibiting Myt1 forced G2/M-arrested cells into an unscheduled transition to the mitotic phase without complete resolution of DNA damage. This forced entry into mitosis, along with persistent DNA damage, resulted in severe mitotic abnormalities. Ultimately, these aberrations led to mitotic exit with substantial apoptosis. Preclinical animal studies demonstrated that the combination regimen involving TMZ and RP-6306 prolonged the overall survival of glioma-bearing mice. CONCLUSIONS: Collectively, our findings highlight the potential of targeting cell cycle timing through Myt1 inhibition as an effective strategy to enhance the efficacy of current standard cancer therapies, potentially leading to improved disease outcomes.
Assuntos
Antineoplásicos Alquilantes , Dano ao DNA , Pontos de Checagem da Fase G2 do Ciclo Celular , Temozolomida , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Animais , Temozolomida/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Camundongos , Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Alquilantes/uso terapêutico , Dano ao DNA/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sistemas CRISPR-Cas , Camundongos Nus , Linhagem Celular Tumoral , Células Tumorais Cultivadas , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Reparo do DNA/efeitos dos fármacosRESUMO
The cell cycle includes two checkpoint arrests allowing to repair of damaged DNA. Many cancer cell lines exhibit weak G1 checkpoint mechanisms relying significantly more on the G2 checkpoint than do healthy cells. Inhibition of Myt1 kinase (PKMYT1), a forgotten member of the Wee family, cyclin-dependent kinase 1 (Cdk1) inhibitory kinase, target for G2 checkpoint abrogation, whose inhibition forces cells into premature unchecked mitosis resulting in cell death, is a promising concept for anticancer therapy. There are not many inhibitors of this emerging, potentially clinically important kinase. Herein, the valuable insight into structural features and binding mechanisms of diaminopyrimidines, aminoquinolines, quinazolines, pyrido[2,3-d]pyrimidines, pyrazolo[3,4-d]pyrimidines, and pyrrolo[2,3-b]quinoxalines, as well as finally made a general scheme of fragmented structures of Myt1 inhibitors with the enzyme, offer potential frameworks useful for future directions, for further chemical optimizations, in the discovery and the design of novel effective structures, potential therapeutics.
Assuntos
Proteínas de Ciclo Celular , Neoplasias , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteína Quinase CDC2/metabolismo , Mitose , Pontos de Checagem da Fase G2 do Ciclo Celular , Pirimidinas/farmacologia , Neoplasias/metabolismo , Fosforilação , Proteínas de Membrana/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Cell cycle checkpoint kinases serve as important therapeutic targets for various cancers. When they are inhibited by small molecules, checkpoint abrogation can induce cell death or further sensitize cancer cells to other genotoxic therapies. Particularly aberrant Cdk1 activation at the G2/M checkpoint by kinase inhibitors causing unscheduled mitotic entry and mitotic arrest was found to lead to DNA damage and cell death selectively in cancer cells. Promising drugs inhibiting kinases like Wee1 (Adavosertib), Wee1+Myt1 (PD166285), ATR (AZD6738) and Chk1 (UCN-01) have been developed, but clinical data has shown variable efficacy for them with poorly understood mechanisms of resistance. Our lab recently identified Myt1 as a predictive biomarker of acquired resistance to the Wee1 kinase inhibitor, Adavosertib. Here, we investigate the role of Myt1 overexpression in promoting resistance to inhibitors (PD166285, UCN-01 and AZD6738) of other kinases regulating cell cycle progression. We demonstrate that Myt1 confers resistance by compensating Cdk1 inhibition in the presence of these different kinase inhibitors. Myt1 overexpression leads to reduced premature mitotic entry and decreased length of mitosis eventually leading to increased survival rates in Adavosertib treated cells. Elevated Myt1 levels also conferred resistance to inhibitors of ATR or Chk1 inhibitor. Our data supports that Myt1 overexpression is a common mechanism by which cancer cells can acquire resistance to a variety of drugs entering the clinic that aim to induce mitotic catastrophe by abrogating the G2/M checkpoint.
RESUMO
The MYT1L gene plays a critical role in brain development, promoting the differentiation and proliferation of cells, important for the formation of brain connections. MYT1L is also involved in regulating the development of the hypothalamus, which is a crucial actor in weight regulation. Genetic variants in the MYT1L are associated with a range of developmental disorders, including intellectual disability, autism spectrum disorder, facial dysmorphisms, and epilepsy. The specific role of MYT1L in epilepsy remains elusive and no patients with developmental and epileptic encephalopathy (DEE) have been described so far. In this study, we report a patient with DEE presenting with severe refractory epilepsy, obesity, and behavioral abnormalities. Exome sequencing led to the identification of the heterozygous variant NM_001303052.2: c.1717G>A, p.(Gly573Arg) (chr2-1910340-C-T; GRCh38.p14) in the MYT1L gene. This variant was found to be inherited by the father, who was a mosaic and did not suffer from any neuropsychiatric disorders. Our observations expand the molecular and phenotype spectrum of MYT1L-related disorders, suggesting that affected individuals may present with severe epileptic phenotype leading to neurocognitive deterioration. Furthermore, we show that mosaic parents may not display the disease phenotype, with relevant implications for genetic counseling.