RESUMO
Glioblastoma stem cells (GSCs) are implicated in tumor neovascularization, invasiveness, and therapeutic resistance. To illuminate mechanisms governing these hallmark features, we developed a de novo glioblastoma multiforme (GBM) model derived from immortalized human neural stem/progenitor cells (hNSCs) to enable precise system-level comparisons of pre-malignant and oncogene-induced malignant states of NSCs. Integrated transcriptomic and epigenomic analyses uncovered a PAX6/DLX5 transcriptional program driving WNT5A-mediated GSC differentiation into endothelial-like cells (GdECs). GdECs recruit existing endothelial cells to promote peritumoral satellite lesions, which serve as a niche supporting the growth of invasive glioma cells away from the primary tumor. Clinical data reveal higher WNT5A and GdECs expression in peritumoral and recurrent GBMs relative to matched intratumoral and primary GBMs, respectively, supporting WNT5A-mediated GSC differentiation and invasive growth in disease recurrence. Thus, the PAX6/DLX5-WNT5A axis governs the diffuse spread of glioma cells throughout the brain parenchyma, contributing to the lethality of GBM.
Assuntos
Glioblastoma/genética , Glioblastoma/patologia , Invasividade Neoplásica/genética , Proteína Wnt-5a/genética , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Epigenômica , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Neurais/metabolismo , Fator de Transcrição PAX6/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The 16-subunit Constitutive Centromere-associated Network (CCAN)-based inner kinetochore is well-known for connecting centromeric chromatin to the spindle-binding outer kinetochore. Here, we report a non-canonical role for the inner kinetochore in directly regulating sister-chromatid cohesion at centromeres. We provide biochemical, X-ray crystal structure, and intracellular ectopic localization evidence that the inner kinetochore directly binds cohesin, a ring-shaped multi-subunit complex that holds sister chromatids together from S-phase until anaphase onset. This interaction is mediated by binding of the 5-subunit CENP-OPQUR sub-complex of CCAN to the Scc1-SA2 sub-complex of cohesin. Mutation in the CENP-U subunit of the CENP-OPQUR complex that abolishes its binding to the composite interface between Scc1 and SA2 weakens centromeric cohesion, leading to premature separation of sister chromatids during delayed metaphase. We further show that CENP-U competes with the cohesin release factor Wapl for binding the interface of Scc1-SA2, and that the cohesion-protecting role for CENP-U can be bypassed by depleting Wapl. Taken together, this study reveals an inner kinetochore-bound pool of cohesin, which strengthens centromeric sister-chromatid cohesion to resist metaphase spindle pulling forces.
Assuntos
Proteínas de Ciclo Celular , Centrômero , Cromátides , Proteínas Cromossômicas não Histona , Cinetocoros , Cinetocoros/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Cromátides/metabolismo , Cromátides/genética , Centrômero/metabolismo , Coesinas , Células HeLa , Ligação Proteica , Cristalografia por Raios XRESUMO
Tumor maintenance relies on continued activity of driver oncogenes, although their rate-limiting role is highly context dependent. Oncogenic Kras mutation is the signature event in pancreatic ductal adenocarcinoma (PDAC), serving a critical role in tumor initiation. Here, an inducible Kras(G12D)-driven PDAC mouse model establishes that advanced PDAC remains strictly dependent on Kras(G12D) expression. Transcriptome and metabolomic analyses indicate that Kras(G12D) serves a vital role in controlling tumor metabolism through stimulation of glucose uptake and channeling of glucose intermediates into the hexosamine biosynthesis and pentose phosphate pathways (PPP). These studies also reveal that oncogenic Kras promotes ribose biogenesis. Unlike canonical models, we demonstrate that Kras(G12D) drives glycolysis intermediates into the nonoxidative PPP, thereby decoupling ribose biogenesis from NADP/NADPH-mediated redox control. Together, this work provides in vivo mechanistic insights into how oncogenic Kras promotes metabolic reprogramming in native tumors and illuminates potential metabolic targets that can be exploited for therapeutic benefit in PDAC.
Assuntos
Adenocarcinoma/metabolismo , Modelos Animais de Doenças , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Humanos , Camundongos , Proteínas Proto-Oncogênicas p21(ras)/genética , Transcrição GênicaRESUMO
The ring-shaped Cohesin complex, consisting of core subunits Smc1, Smc3, Scc1, and SA2 (or its paralog SA1), topologically entraps two duplicated sister DNA molecules to establish sister chromatid cohesion in S-phase. It remains largely elusive how the Cohesin release factor Wapl binds the Cohesin complex, thereby inducing Cohesin disassociation from mitotic chromosomes to allow proper resolution and separation of sister chromatids. Here, we show that Wapl uses two structural modules containing the FGF motif and the YNARHWN motif, respectively, to simultaneously bind distinct pockets in the extensive composite interface between Scc1 and SA2. Strikingly, only when both docking modules are mutated, Wapl completely loses the ability to bind the Scc1-SA2 interface and release Cohesin, leading to erroneous chromosome segregation in mitosis. Surprisingly, Sororin, which contains a conserved FGF motif and functions as a master antagonist of Wapl in S-phase and G2-phase, does not bind the Scc1-SA2 interface. Moreover, Sgo1, the major protector of Cohesin at mitotic centromeres, can only compete with the FGF motif but not the YNARHWN motif of Wapl for binding Scc1-SA2 interface. Our data uncover the molecular mechanism by which Wapl binds Cohesin to ensure precise chromosome segregation.
Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Segregação de Cromossomos , Coesinas , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Humanos , Ligação Proteica , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Motivos de Aminoácidos , Mitose , Cromátides/metabolismo , Proteínas de Transporte , Proteínas Proto-OncogênicasRESUMO
Chromosome segregation in mitosis requires the removal of catenation between sister chromatids. Timely decatenation of sister DNAs at mitotic centromeres by topoisomerase IIα (TOP2A) is crucial to maintain genomic stability. The chromatin factors that recruit TOP2A to centromeres during mitosis remain unknown. Here, we show that histone H2A Thr-120 phosphorylation (H2ApT120), a modification generated by the mitotic kinase Bub1, is necessary and sufficient for the centromeric localization of TOP2A. Phosphorylation at residue-120 enhances histone H2A binding to TOP2A in vitro. The C-gate and the extreme C-terminal region are important for H2ApT120-dependent localization of TOP2A at centromeres. Preventing H2ApT120-mediated accumulation of TOP2A at mitotic centromeres interferes with sister chromatid disjunction, as evidenced by increased frequency of anaphase ultra-fine bridges (UFBs) that contain catenated DNA. Tethering TOP2A to centromeres bypasses the requirement for H2ApT120 in suppressing anaphase UFBs. These results demonstrate that H2ApT120 acts as a landmark that recruits TOP2A to mitotic centromeres to decatenate sister DNAs. Our study reveals a fundamental role for histone phosphorylation in resolving centromere DNA entanglements and safeguarding genomic stability during mitosis.
Assuntos
Centrômero/metabolismo , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/metabolismo , DNA/metabolismo , Histonas/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/química , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Sítios de Ligação , Linhagem Celular , Segregação de Cromossomos , Instabilidade Genômica , Células HeLa , Humanos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , TreoninaRESUMO
Replication fork reversal occurs via a two-step process that entails reversal initiation and reversal extension. DNA topoisomerase IIalpha (TOP2A) facilitates extensive fork reversal, on one hand through resolving the topological stress generated by the initial reversal, on the other hand via its role in recruiting the SUMO-targeted DNA translocase PICH to stalled forks in a manner that is dependent on its SUMOylation by the SUMO E3 ligase ZATT. However, how TOP2A activities at stalled forks are precisely regulated remains poorly understood. Here we show that, upon replication stress, the SUMO-targeted ubiquitin E3 ligase RNF4 accumulates at stalled forks and targets SUMOylated TOP2A for ubiquitination and degradation. Downregulation of RNF4 resulted in aberrant activation of the ZATT-TOP2A-PICH complex at stalled forks, which in turn led to excessive reversal and elevated frequencies of fork collapse. These results uncover a previously unidentified regulatory mechanism that regulates TOP2A activities at stalled forks and thus the extent of fork reversal.
Assuntos
Replicação do DNA , Instabilidade Genômica , Replicação do DNA/genética , Instabilidade Genômica/genética , Humanos , Proteínas Nucleares/metabolismo , Sumoilação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Posttranslational modifications (PTMs) of viral proteins play critical roles in virus infection. The role of neddylation in enterovirus 71 (EV71) replication remains poorly defined. Here, we showed that the structural protein VP2 of EV71 can be modified by neural precursor cell-expressed developmentally downregulated protein 8 (NEDD8) in an E3 ligase X-linked inhibitor of apoptosis protein (XIAP)-dependent manner. Mutagenesis and biochemical analyses mapped the neddylation site at lysine 69 (K69) of VP2 and demonstrated that neddylation reduced the stability of VP2. In agreement with the essential role of VP2 in viral replication, studies with EV71 reporter viruses with wild-type VP2 (enhanced green fluorescent protein [EGFP]-EV71) and a K69R mutant VP2 (EGFP-EV71-VP2 K69R) showed that abolishment of VP2 neddylation increased EV71 replication. In support of this finding, overexpression of NEDD8 significantly inhibited the replication of wild-type EV71 and EGFP-EV71, but not EGFP-EV71-VP2 K69R, whereas pharmacologic inhibition of neddylation with the NEDD8-activating enzyme inhibitor MLN4924 promoted the replication of EV71 in biologically relevant cell types. Our results thus support the notion that EV71 replication can be negatively regulated by host cellular and pathobiological cues through neddylation of VP2 protein. IMPORTANCE Neddylation is a ubiquitin-like posttranslational modification by conjugation of neural precursor cell-expressed developmentally downregulated protein 8 (NEDD8) to specific proteins for regulation of their metabolism and biological activities. In this study, we demonstrated for the first time that EV71 VP2 protein is neddylated at K69 residue to promote viral protein degradation and consequentially suppress multiplication of the virus. Our findings advance knowledge related to the roles of VP2 in EV71 virulence and the neddylation pathway in the host restriction of EV71 infection.
Assuntos
Proteínas do Capsídeo , Enterovirus Humano A , Processamento de Proteína Pós-Traducional , Replicação Viral , Animais , Proteínas do Capsídeo/química , Linhagem Celular Tumoral , Chlorocebus aethiops , Enterovirus Humano A/fisiologia , Células HEK293 , Humanos , Proteína NEDD8/metabolismo , Células Vero , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
BACKGROUND: Ischemia/reperfusion (I/R) is a pathological process that causes severe damage. Propofol is known to alleviate I/R-related injury; however, the exact function and underlying mechanisms are not fully understood. METHODS: Using an oxygen glucose deprivation/re-oxygenation (OGD/R) method, an in vitro I/R injury model was induced. The cell viability and the level of Fe2+, glutathione synthetase (GSH), and malondialdehyde (MDA) were evaluated using kits. Luciferase reporter gene assay, chromatin immunoprecipitation, and RNA immunoprecipitation (RIP) were used to verify the interaction between molecules. The m6A level of BECN1 mRNA was determined through methylated RIP. RESULTS: Propofol-treated OGD/R models showed reduced levels of Fe2+ and MDA, while the cell viability and the level of GSH increased. Propofol inhibited ferroptosis by down-regulating HIF-1α in OGD/R-treated HT22 cells. HIF-1α is bound to the promoter region of YTHDF1 to promote its transcription, and YTHDF1 promoted ferroptosis by stabilizing the mRNA of BECN1. The suppressive effect of propofol on OGD/R-induced ferroptosis was reversed by the overexpression of YTHDF1. CONCLUSIONS: Our study revealed that the HIF-1α/YTHDF1/BECN1 axis in OGD/R-treated HT22 cells promotes ferroptosis, and administration of propofol can inhibit this axis to avoid cell death. This study provides a novel insight for the neuroprotective function of propofol.
Assuntos
Ferroptose , Propofol , Humanos , Propofol/farmacologia , Propofol/uso terapêutico , Neurônios , Morte Celular , Glucose , Proteína Beclina-1 , Proteínas de Ligação a RNARESUMO
The current systematic analysis and meta-analysis was aimed to evaluate the association between radiation-induced lymphopenia (RIL) and survival of women with cervical cancer (CC). PubMed, Embase, Web of Science, and Cochrane Library were searched for relevant cohort studies comparing survival between women with CC who developed versus not developed RIL after radiotherapy. We pooled the results using a random-effects model that incorporates heterogeneity. In the meta-analysis, 952 women with CC were included from eight cohort studies. Overall, 378 (39.7%) of them had RIL after radiotherapy. During a median follow-up duration of 41.8 months, pooled results showed that RIL was independently associated with poor overall survival (hazard ratio [HR]: 2.67, 95% confidence interval [CI]: 1.81 to 3.94, p < 0.001; I2 = 20%) and progression-free survival (HR: 2.17, 95% CI: 1.58 to 2.98, p < 0.001; I2 = 0%). Predefined subgroup analyses showed similar results in patients with grade 3-4 and grade 4 RIL, in patients with RIL diagnosed during or after the radiotherapy, and in studies with quality score of seven or eight points (p values for subgroup effect all < 0.05). In conclusion, women with RIL were associated with poor survival after radiotherapy for CC.
Assuntos
Linfopenia , Neoplasias do Colo do Útero , Humanos , Feminino , Neoplasias do Colo do Útero/radioterapia , Linfopenia/etiologia , Modelos de Riscos Proporcionais , Intervalo Livre de DoençaRESUMO
BACKGROUND: ß-Amyloid (Aß) protein is a pivotal pathogenetic factor in Alzheimer's disease (AD). However, increasing evidence suggests that the brain has to continuously produce excessive Aß to efficaciously prevent pathogenic micro-organism infections, which induces and accelerates the disease process of AD. Meanwhile, Aß exhibits activity against herpes simplex virus type 1 (HSV-1) and influenza A virus (IAV) replication, but not against other neurotropic viruses. Enterovirus A71 (EV-A71) is the most important neurotropic enterovirus in the post-polio era. Given the limitation of existing research on the relationship between Aß and other virus infections, this study aimed to investigate the potent activity of Aß on EV-A71 infection and extended the potential function of Aß in other unenveloped viruses may be linked to Alzheimer's disease or infectious neurological diseases. METHODS: Aß peptides 1-42 are a major pathological factor of senile plaques in Alzheimer's disease (AD). Thus, we utilized Aß1-42 as a test subject to perform our study. The production of monomer Aß1-42 and their high-molecular oligomer accumulations in neural cells were detected by immunofluorescence assay, ELISA, or Western blot assay. The inhibitory activity of Aß1-42 peptides against EV-A71 in vitro was detected by Western blot analysis or qRT-PCR. The mechanism of Aß1-42 against EV-A71 replication was analyzed by time-of-addition assay, attachment inhibition assay, pre-attachment inhibition analysis, viral-penetration inhibition assay, TEM analysis of virus agglutination, and pull-down assay. RESULTS: We found that EV-A71 infection induced Aß production and accumulation in SH-SY5Y cells. We also revealed for the first time that Aß1-42 efficiently inhibited the RNA level of EV-A71 VP1, and the protein levels of VP1, VP2, and nonstructural protein 3AB in SH-SY5Y, Vero, and human rhabdomyosarcoma (RD) cells. Mechanistically, we demonstrated that Aß1-42 primarily targeted the early stage of EV-A71 entry to inhibit virus replication by binding virus capsid protein VP1 or scavenger receptor class B member 2. Moreover, Aß1-42 formed non-enveloped EV-A71 particle aggregates within a certain period and bound to the capsid protein VP1, which partially caused Aß1-42 to prevent viruses from infecting cells. CONCLUSIONS: Our findings unveiled that Aß1-42 effectively inhibited nonenveloped EV-A71 by targeting the early phase of an EV-A71 life cycle, thereby extending the potential function of Aß in other non-envelope viruses linked to infectious neurological diseases.
Assuntos
Doença de Alzheimer , Enterovirus Humano A , Infecções por Enterovirus , Enterovirus , Neuroblastoma , Peptídeos beta-Amiloides , Antígenos Virais/genética , Proteínas do Capsídeo/genética , Enterovirus/genética , Enterovirus Humano A/genética , Humanos , Fragmentos de Peptídeos , RNA , Receptores Depuradores/metabolismoRESUMO
OBJECTIVE: For treatment of infected femoral artery pseudoaneurysms (IFAPs) with the covered stent, debridement technique is important but frequently ignored. Our study aims to review our experience and outcomes of patients undergoing covered stents placement and debridement with vacuum sealing drainage (VSD). METHODS: This study retrospectively analyzed 41 intravenous drug addicts with IFAP who received covered stent implantation and debridement with VSD from January 2015 to December 2020. The diagnosis was based on the previous history of local injection and the presence of pulsatile mass at the injection site. All cases were confirmed by CT angiography (CTA), ultrasound, or digital subtraction angiography (DSA). Technical success, time of wound care, and clinical outcomes were evaluated. RESULTS: Technical success was achieved in all patients. The interval from diagnosis to treatment was 26 ± 11 hours. The time of continuous drainage with VSD was 18.79 ± 6.56 days. 38 patients (92.68%) with fresh granulation tissue were sutured and discharged from the hospital. Stents in 31(91.18%) of 34 cases were patent during follow-ups. Three patients had stent occlusion caused by thrombosis, and two of them were complicated with stent infection. The two infectious stents were removed and the femoral arteries were ligated. One of them received open-surgical reconstruction with the great saphenous because of claudication. Two patients were admitted to the hospital for rebleeding caused by drug abuse relapse. CONCLUSIONS: Covered stents placement is convenient and rapid to control massive hemorrhage in IFAPs of intravenous drug abuse. Early debridement of infected tissue with continued VSD may shorten the time of wound care and make the incidence of stent infection relatively low. Meanwhile, the patency in a short time follow-up is acceptable. These results indicate that covered stents implantation with VSD may be a safe, effective, and feasible measure for the treatment of IFAPs.
Assuntos
Falso Aneurisma , Usuários de Drogas , Tratamento de Ferimentos com Pressão Negativa , Falso Aneurisma/diagnóstico por imagem , Falso Aneurisma/etiologia , Falso Aneurisma/terapia , Desbridamento/efeitos adversos , Drenagem/efeitos adversos , Humanos , Tratamento de Ferimentos com Pressão Negativa/efeitos adversos , Estudos Retrospectivos , Stents/efeitos adversos , Resultado do TratamentoRESUMO
BACKGROUND: Large-scale data on esophagogastroduodenoscopy (EGD) in China are scarce. This study aimed to assess the indications and diagnostic yield of EGD in children and the relationship between factors (such as age, sex, and indications) and diagnostic yield. METHODS: We performed a prospective cross-sectional observational study involving patients aged < 18 years who underwent diagnostic EGD. The study was conducted in five children's hospitals, each in a different city. Demographic features, indications for endoscopy, and endoscopic and histopathological findings were collected. Univariable and multivariable ordinal logistic regression analyses of the relationship between the factors and diagnostic yield were performed. RESULTS: The study included 2268 patients (male/female ratio, 1.3:1) with a median age of 8.68 years. Among the 2268 children, the most frequent indications were abdominal pain in 1954 (86.2%), recurrent vomiting in 706 (31.1%), weight loss in 343 (15.1%), and others. The endoscopic yield was 62.5% and was the highest in patients with dysphagia (90.9%). The histologic yield was 30.4% and was the highest in patients with unexplained anemia (45.5%). On multivariable regression analysis, the endoscopic yield was associated with dysphagia, gastrointestinal (GI) bleeding, and recurrent vomiting, and the histologic yield was associated with age. Different groups of patients with abdominal pain had variable probabilities of abnormal endoscopic findings. CONCLUSIONS: The most frequent indication of pediatric EGD is abdominal pain, with variable probabilities of abnormal endoscopic findings in different groups. Endoscopic yield and histologic yield are associated with certain alarming features. TRIAL REGISTRATION: The trial registration number (ClinicalTrials. gov): NCT03603093 (The study was registered on 27/07/2018).
Assuntos
Transtornos de Deglutição , Dor Abdominal/diagnóstico , Criança , China , Estudos Transversais , Endoscopia Gastrointestinal , Feminino , Hemorragia Gastrointestinal/diagnóstico , Humanos , Masculino , Estudos Prospectivos , Estudos Retrospectivos , VômitoRESUMO
Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, and there is no specific drug to treat it. Recent results showed that 17-beta-hydroxysteroid dehydrogenase type 13 (HSD17B13) is associated with liver diseases, but these conclusions are controversial. Here, we showed that HSD17B13 was more highly expressed in the livers of NAFLD patients, and high expression was induced in the livers of murine NAFLD models and cultural hepatocytes treated using various etiologies. The high HSD17B13 expression in the hepatocytes facilitated the progression of NAFLD by directly stabilizing the intracellular lipid drops and by indirectly activating hepatic stellate cells. When HSD17B13 was overexpressed in the liver, it aggravated liver steatosis and fibrosis in mice fed with a high-fat diet, while down-regulated the high expression of HSD17B13 by short hairpin RNAs produced a therapeutic effect in the NAFLD mice. We concluded that high HSD17B13 expression is a good target for the development of drugs to treat NAFLD.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Animais , Dieta Hiperlipídica/efeitos adversos , Células Estreladas do Fígado/metabolismo , Hepatócitos/metabolismo , Humanos , Camundongos , Hepatopatia Gordurosa não Alcoólica/metabolismoRESUMO
Chronic hepatitis induced by hepatitis B virus (HBV) infection is a serious public health problem, leading to hepatic cirrhosis and liver cancer. Although the currently approved medications can reliably decrease the virus load and prevent the development of hepatic diseases, they fail to induce durable off-drug control of HBV replication in the majority of patients. The roots of Isatis indigotica Fortune ex Lindl., a traditional Chinese medicine, were frequently used for the prevention of viral disease in China. In the present study, (-)-lariciresinol ((-)-LRSL), isolated from the roots of Isatis indigotica Fortune ex Lindl., was found to inhibit HBV DNA replication of both wild-type and nucleos(t)ide analogues (NUCs)-resistant strains in vitro. Mechanism studies revealed that (-)-LRSL could block RNA production after treatment, followed by viral proteins, and then viral particles and DNA. Promoter reporter assays and RNA decaying dynamic experiments indicated that (-)-LRSL mediated HBV RNA reduction was mainly due to transcriptional inhibition rather than degradation. Moreover, (-)-LRSL in a dose-dependent manner also inhibited other animal hepadnaviruses, including woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV). Combining the analysis of RNA-seq, we further found that the decrease in HBV transcriptional activity by (-)-LRSL may be related to hepatocyte nuclear factor 1α (HNF1α). Taken together, (-)-LRSL represents a novel chemical entity that inhibits HBV replication by regulating HNF1α mediated HBV transcription, which may provide a new perspective for HBV therapeutics.
Assuntos
Vírus da Hepatite B , Isatis , Animais , Furanos , Vírus da Hepatite B/metabolismo , Humanos , Isatis/genética , Lignanas , RNA/metabolismo , Transcrição ViralRESUMO
This study explored the anticoagulant material basis and mechanism of Trichosanthis Semen and its shell and kernel based on spectrum-effect relationship-integrated molecular docking. High performance liquid chromatography(HPLC) fingerprints of Trichosanthis Semen and its shell and kernel were established. Prothrombin time(PT) and activated partial thromboplastin time(APTT) in mice in the low-and high-dose(5, 30 g·kg~(-1), respectively) Trichosanthis Semen, the shell, and kernel groups were determined as the coagulation markers. The spectrum-effect relationship and anticoagulant material basis of Trichosanthis Semen and its shell and kernel were analyzed with mean value calculation method of Deng's correlation degree(MATLAB) and the common effective component cluster was obtained. Then the common targets of the component cluster and coagulation were retrieved from TCMSP, Swiss-TargetPrediction, GenCLiP3, GeneCards, and DAVID, followed by Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment of the targets. The main anticoagulant molecular mechanism of the component cluster was verified by SYBYL-X 2.1.1. The spectrum-effect relationship of Trichosanthis Semen and its shell and kernel was in positive correlation with the dosage. The contribution of each component to anticoagulation was not the same, suggesting that the material basis for anticoagulation was different, but they have common effective components(i.e. common material basis), such as adenine(peak 3), uracil(peak 4), hypoxanthine(peak 6), xanthine(peak 9), and adenosine(peak 11). Network pharmacology showed that these components can act on multiple target proteins such as NOS3, KDR, and PTGS2, and exert anticoagulant effect through multiple pathways such as VEGF signaling pathway. They involved the biological functions such as proteolysis, cell component such as cytosol, and molecular functions. The results of molecular docking showed that the binding free energy of these components with NOS3(PDB ID: 1 D0 C), KDR(PDB ID: 5 AMN), and PTGS2(PDB ID: 4 COX) was ≤-5 kJ·mol~(-1), and the docking conformations were stable. Spectrum-effect relationship-integrated molecular docking can be used for the optimization, virtual screening, and verification of complex chemical and biological information of Chinese medicine. Trichosanthis Semen and its shell and kernel have the common material basis for anticoagulation and they exert the anticoagulant through multiple targets and pathways.
Assuntos
Anticoagulantes , Medicamentos de Ervas Chinesas , Animais , Anticoagulantes/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Ontologia Genética , Camundongos , Simulação de Acoplamento Molecular , SêmenRESUMO
Transforming growth factor beta (TGF-ß) plays an important role in the viral liver disease progression via controlling viral propagation and mediating inflammation-associated responses. However, the antiviral activities and mechanisms of TGF-ß isoforms, including TGF-ß1, TGF-ß2 and TGF-ß3, remain unclear. Here, we demonstrated that all of the three TGF-ß isoforms were increased in Huh7.5 cells infected by hepatitis C virus (HCV), but in turn, the elevated TGF-ß isoforms could inhibit HCV propagation with different potency in infectious HCV cell culture system. TGF-ß isoforms suppressed HCV propagation through interrupting several different stages in the whole HCV life cycle, including virus entry and intracellular replication, in TGF-ß/SMAD signalling pathway-dependent and TGF-ß/SMAD signalling pathway-independent manners. TGF-ß isoforms showed additional anti-HCV activities when combined with each other. However, the elevated TGF-ß1 and TGF-ß2, not TGF-ß3, could also induce liver fibrosis with a high expression of type I collagen alpha-1 and α-smooth muscle actin in LX-2 cells. Our results showed a new insight into TGF-ß isoforms in the HCV-related liver disease progression.
Assuntos
Hepacivirus/efeitos dos fármacos , Hepacivirus/crescimento & desenvolvimento , Hepatite C/virologia , Transdução de Sinais , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Sequência de Aminoácidos , Antivirais/farmacologia , Linhagem Celular Tumoral , Hepatite C/patologia , Humanos , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacologia , RNA Viral , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Fator de Crescimento Transformador beta2/metabolismo , Fator de Crescimento Transformador beta2/farmacologia , Fator de Crescimento Transformador beta3/metabolismo , Fator de Crescimento Transformador beta3/farmacologia , Internalização do Vírus/efeitos dos fármacosRESUMO
The bromodomain-containing protein 7 (BRD7) is a tumour suppressor protein with critical roles in cell cycle transition and transcriptional regulation. Whether BRD7 is regulated by post-translational modifications remains poorly understood. Here, we find that chemotherapy-induced DNA damage leads to the rapid degradation of BRD7 in various cancer cell lines. PARP-1 binds and poly(ADP)ribosylates BRD7, which enhances its ubiquitination and degradation through the PAR-binding E3 ubiquitin ligase RNF146. Moreover, the PARP1 inhibitor Olaparib significantly enhances the sensitivity of BRD7-positive cancer cells to chemotherapeutic drugs, while it has little effect on cells with low BRD7 expression. Taken together, our findings show that PARP1 induces the degradation of BRD7 resulting in cancer cell resistance to DNA-damaging agents. BRD7 might thus serve as potential biomarker in clinical trial for the prediction of synergistic effects between chemotherapeutic drugs and PARP inhibitors.
Assuntos
Antineoplásicos/farmacologia , Proteínas Cromossômicas não Histona/metabolismo , Dano ao DNA/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli ADP Ribosilação/efeitos dos fármacos , Células A549 , Linhagem Celular , Linhagem Celular Tumoral , DNA/metabolismo , Reparo do DNA/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
Miyoshi myopathy (MM) is a rare autosomal recessive disorder caused by dysferlin (DYSF) gene mutation. Miyoshi myopathy-inducing mutation sites in the DYSF gene have been discovered worldwide. In the present study, a patient with progressive lower extremity weakness is reported, for which MM was diagnosed according to clinical manifestations, muscle biopsy, and immunohistochemistry. In addition, the DYSF gene of the patient and his parents was sequenced and analyzed and two heterozygous mutations of the DYSF gene (c.4756C> T and c.5316dupC) were discovered. The first mutation correlated with MM while the second was a new mutation. The patient was diagnosed with a compound heterozygous mutation. The mutation site is a new member of pathogenic MM gene mutations.
Assuntos
Proteínas de Membrana , Proteínas Musculares , Alelos , Miopatias Distais , Disferlina/genética , Humanos , Proteínas de Membrana/genética , Proteínas Musculares/genética , Atrofia Muscular , Mutação/genéticaRESUMO
The chromosomal passenger complex (CPC) is a master regulator of mitosis. CPC consists of inner centromere protein (INCENP), Survivin, Borealin, and the kinase Aurora B and plays key roles in regulating kinetochore-microtubule attachments and spindle assembly checkpoint signaling. However, the role of CPC in sister chromatid cohesion, mediated by the cohesin complex, remains incompletely understood. Here, we show that Aurora B kinase activity contributes to centromeric cohesion protection partly through promoting kinetochore localization of the kinase Bub1. Interestingly, disrupting the interaction of INCENP with heterochromatin protein 1 (HP1) in HeLa cells selectively weakens cohesion at mitotic centromeres without detectably reducing the kinase activity of Aurora B. Thus, through this INCENP-HP1 interaction, the CPC also protects centromeric cohesion independently of Aurora B kinase activity. Moreover, the requirement for the INCENP-HP1 interaction in centromeric cohesion protection can be bypassed by tethering HP1 to centromeres or by depleting the cohesin release factor Wapl. We provide further evidence suggesting that the INCENP-HP1 interaction protects centromeric cohesion by promoting the centromere localization of Haspin, a protein kinase that antagonizes Wapl activity at centromeres. Taken together, this study identifies Aurora B kinase activity-dependent and -independent roles for the CPC in regulating centromeric cohesion during mitosis in human cells.
Assuntos
Aurora Quinase B/metabolismo , Centrômero/metabolismo , Cromátides/metabolismo , Mitose/fisiologia , Complexos Multiproteicos/metabolismo , Aurora Quinase B/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Centrômero/genética , Cromátides/genética , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismoRESUMO
The inner centromere region of a mitotic chromosome critically regulates sister chromatid cohesion and kinetochore-microtubule attachments. However, the molecular mechanism underlying inner centromere assembly remains elusive. Here, using CRISPR/Cas9-based gene editing in HeLa cells, we disrupted the interaction of Shugoshin 1 (Sgo1) with histone H2A phosphorylated on Thr-120 (H2ApT120) to selectively release Sgo1 from mitotic centromeres. Interestingly, cells expressing the H2ApT120-binding defective mutant of Sgo1 have an elevated rate of chromosome missegregation accompanied by weakened centromeric cohesion and decreased centromere accumulation of the chromosomal passenger complex (CPC), an integral part of the inner centromere and a key player in the correction of erroneous kinetochore-microtubule attachments. When artificially tethered to centromeres, a Sgo1 mutant defective in binding protein phosphatase 2A (PP2A) is not able to support proper centromeric cohesion and CPC accumulation, indicating that the Sgo1-PP2A interaction is essential for the integrity of mitotic centromeres. We further provide evidence indicating that Sgo1 protects centromeric cohesin to create a binding site for the histone H3-associated protein kinase Haspin, which not only inhibits the cohesin release factor Wapl and thereby strengthens centromeric cohesion but also phosphorylates histone H3 at Thr-3 to position CPC at inner centromeres. Taken together, our findings reveal a positive feedback-based mechanism that ensures proper assembly of the functional inner centromere during mitosis. They further suggest a causal link between centromeric cohesion defects and chromosomal instability in cancer cells.