RESUMO
Receptor tyrosine kinase (RTK)-mediated activation of downstream effector pathways such as the RAS GTPase/MAP kinase (MAPK) signaling cascade is thought to occur exclusively from lipid membrane compartments in mammalian cells. Here, we uncover a membraneless, protein granule-based subcellular structure that can organize RTK/RAS/MAPK signaling in cancer. Chimeric (fusion) oncoproteins involving certain RTKs including ALK and RET undergo de novo higher-order assembly into membraneless cytoplasmic protein granules that actively signal. These pathogenic biomolecular condensates locally concentrate the RAS activating complex GRB2/SOS1 and activate RAS in a lipid membrane-independent manner. RTK protein granule formation is critical for oncogenic RAS/MAPK signaling output in these cells. We identify a set of protein granule components and establish structural rules that define the formation of membraneless protein granules by RTK oncoproteins. Our findings reveal membraneless, higher-order cytoplasmic protein assembly as a distinct subcellular platform for organizing oncogenic RTK and RAS signaling.
Assuntos
Condensados Biomoleculares/metabolismo , Grânulos Citoplasmáticos/metabolismo , Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas ras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ativação Enzimática , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/metabolismo , Células HEK293 , Humanos , Proteína SOS1/metabolismo , Transdução de SinaisRESUMO
Formation of co-transcriptional R-loops underlies replication fork stalling upon head-on transcription-replication encounters. Here, we demonstrate that RAD51-dependent replication fork reversal induced by R-loops is followed by the restart of semiconservative DNA replication mediated by RECQ1 and RECQ5 helicases, MUS81/EME1 endonuclease, RAD52 strand-annealing factor, the DNA ligase IV (LIG4)/XRCC4 complex, and the non-catalytic subunit of DNA polymerase δ, POLD3. RECQ5 disrupts RAD51 filaments assembled on stalled forks after RECQ1-mediated reverse branch migration, preventing a new round of fork reversal and facilitating fork cleavage by MUS81/EME1. MUS81-dependent DNA breaks accumulate in cells lacking RAD52 or LIG4 upon induction of R-loop formation, suggesting that RAD52 acts in concert with LIG4/XRCC4 to catalyze fork religation, thereby mediating replication restart. The resumption of DNA synthesis after R-loop-associated fork stalling also requires active transcription, the restoration of which depends on MUS81, RAD52, LIG4, and the transcription elongation factor ELL. These findings provide mechanistic insights into transcription-replication conflict resolution.
Assuntos
Replicação do DNA/fisiologia , Estruturas R-Loop/genética , Rad51 Recombinase/metabolismo , Linhagem Celular Tumoral , DNA Ligases/metabolismo , DNA Polimerase III/metabolismo , Replicação do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Células HeLa , Humanos , Estruturas R-Loop/fisiologia , Rad51 Recombinase/genética , Rad51 Recombinase/fisiologia , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , RecQ Helicases/metabolismo , RecQ Helicases/fisiologia , Transcrição Gênica/genéticaRESUMO
Cellular mechanisms that safeguard genome integrity are often subverted in cancer. To identify cancer-related genome caretakers, we employed a convergent multi-screening strategy coupled to quantitative image-based cytometry and ranked candidate genes according to multivariate readouts reflecting viability, proliferative capacity, replisome integrity, and DNA damage signaling. This unveiled regulators of replication stress resilience, including components of the pre-mRNA cleavage and polyadenylation complex. We show that deregulation of pre-mRNA cleavage impairs replication fork speed and leads to excessive origin activity, rendering cells highly dependent on ATR function. While excessive formation of RNA:DNA hybrids under these conditions was tightly associated with replication-stress-induced DNA damage, inhibition of transcription rescued fork speed, origin activation, and alleviated replication catastrophe. Uncoupling of pre-mRNA cleavage from co-transcriptional processing and export also protected cells from replication-stress-associated DNA damage, suggesting that pre-mRNA cleavage provides a mechanism to efficiently release nascent transcripts and thereby prevent gene gating-associated genomic instability.
Assuntos
Dano ao DNA , Replicação do DNA , Instabilidade Genômica , Neoplasias/genética , Clivagem do RNA , Precursores de RNA/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Transporte Ativo do Núcleo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Poliadenilação , Precursores de RNA/biossíntese , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Proteínas de Ligação a RNARESUMO
The MUS81-EME1 endonuclease cleaves late replication intermediates at common fragile sites (CFSs) during early mitosis to trigger DNA-repair synthesis that ensures faithful chromosome segregation. Here, we show that these DNA transactions are promoted by RECQ5 DNA helicase in a manner dependent on its Ser727 phosphorylation by CDK1. Upon replication stress, RECQ5 associates with CFSs in early mitosis through its physical interaction with MUS81 and promotes MUS81-dependent mitotic DNA synthesis. RECQ5 depletion or mutational inactivation of its ATP-binding site, RAD51-interacting domain, or phosphorylation site causes excessive binding of RAD51 to CFS loci and impairs CFS expression. This leads to defective chromosome segregation and accumulation of CFS-associated DNA damage in G1 cells. Biochemically, RECQ5 alleviates the inhibitory effect of RAD51 on 3'-flap DNA cleavage by MUS81-EME1 through its RAD51 filament disruption activity. These data suggest that RECQ5 removes RAD51 filaments stabilizing stalled replication forks at CFSs and hence facilitates CFS cleavage by MUS81-EME1.
Assuntos
Sítios Frágeis do Cromossomo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , DNA/biossíntese , Endonucleases/metabolismo , Mitose , RecQ Helicases/metabolismo , Origem de Replicação , Sítios de Ligação , Proteína Quinase CDC2 , Instabilidade Cromossômica , Segregação de Cromossomos , Quinases Ciclina-Dependentes/metabolismo , DNA/genética , Dano ao DNA , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/metabolismo , Endonucleases/genética , Células HEK293 , Células HeLa , Humanos , Fosforilação , Ligação Proteica , Interferência de RNA , Rad51 Recombinase/metabolismo , RecQ Helicases/genética , Fatores de Tempo , TransfecçãoRESUMO
Rationale: Cardiovascular events after chronic obstructive pulmonary disease (COPD) exacerbations are recognized. Studies to date have been post hoc analyses of trials, did not differentiate exacerbation severity, included death in the cardiovascular outcome, or had insufficient power to explore individual outcomes temporally.Objectives: We explore temporal relationships between moderate and severe exacerbations and incident, nonfatal hospitalized cardiovascular events in a primary care-derived COPD cohort.Methods: We included people with COPD in England from 2014 to 2020, from the Clinical Practice Research Datalink Aurum primary care database. The index date was the date of first COPD exacerbation or, for those without exacerbations, date upon eligibility. We determined composite and individual cardiovascular events (acute coronary syndrome, arrhythmia, heart failure, ischemic stroke, and pulmonary hypertension) from linked hospital data. Adjusted Cox regression models were used to estimate average and time-stratified adjusted hazard ratios (aHRs).Measurements and Main Results: Among 213,466 patients, 146,448 (68.6%) had any exacerbation; 119,124 (55.8%) had moderate exacerbations, and 27,324 (12.8%) had severe exacerbations. A total of 40,773 cardiovascular events were recorded. There was an immediate period of cardiovascular relative rate after any exacerbation (1-14 d; aHR, 3.19 [95% confidence interval (CI), 2.71-3.76]), followed by progressively declining yet maintained effects, elevated after one year (aHR, 1.84 [95% CI, 1.78-1.91]). Hazard ratios were highest 1-14 days after severe exacerbations (aHR, 14.5 [95% CI, 12.2-17.3]) but highest 14-30 days after moderate exacerbations (aHR, 1.94 [95% CI, 1.63-2.31]). Cardiovascular outcomes with the greatest two-week effects after a severe exacerbation were arrhythmia (aHR, 12.7 [95% CI, 10.3-15.7]) and heart failure (aHR, 8.31 [95% CI, 6.79-10.2]).Conclusions: Cardiovascular events after moderate COPD exacerbations occur slightly later than after severe exacerbations; heightened relative rates remain beyond one year irrespective of severity. The period immediately after an exacerbation presents a critical opportunity for clinical intervention and treatment optimization to prevent future cardiovascular events.
Assuntos
Doenças Cardiovasculares , Insuficiência Cardíaca , Doença Pulmonar Obstrutiva Crônica , Humanos , Progressão da Doença , Doença Pulmonar Obstrutiva Crônica/complicações , Doença Pulmonar Obstrutiva Crônica/epidemiologia , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Arritmias Cardíacas , Insuficiência Cardíaca/epidemiologia , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/etiologiaRESUMO
BACKGROUND: An immediate, temporal risk of heart failure and arrhythmias after a Chronic Obstructive Pulmonary Disease (COPD) exacerbation has been demonstrated, particularly in the first month post-exacerbation. However, the clinical profile of patients who develop heart failure (HF) or atrial fibrillation/flutter (AF) following exacerbation is unclear. Therefore we examined factors associated with people being hospitalized for HF or AF, respectively, following a COPD exacerbation. METHODS: We conducted two nested case-control studies, using primary care electronic healthcare records from the Clinical Practice Research Datalink Aurum linked to Hospital Episode Statistics, Office for National Statistics for mortality, and socioeconomic data (2014-2020). Cases had hospitalization for HF or AF within 30 days of a COPD exacerbation, with controls matched by GP practice (HF 2:1;AF 3:1). We used conditional logistic regression to explore demographic and clinical factors associated with HF and AF hospitalization. RESULTS: Odds of HF hospitalization (1,569 cases, 3,138 controls) increased with age, type II diabetes, obesity, HF and arrhythmia history, exacerbation severity (hospitalization), most cardiovascular medications, GOLD airflow obstruction, MRC dyspnea score, and chronic kidney disease. Strongest associations were for severe exacerbations (adjusted odds ratio (aOR)=6.25, 95%CI 5.10-7.66), prior HF (aOR=2.57, 95%CI 1.73-3.83), age≥80 years (aOR=2.41, 95%CI 1.88-3.09), and prior diuretics prescription (aOR=2.81, 95%CI 2.29-3.45). Odds of AF hospitalization (841 cases, 2,523 controls) increased with age, male sex, severe exacerbation, arrhythmia and pulmonary hypertension history and most cardiovascular medications. Strongest associations were for severe exacerbations (aOR=5.78, 95%CI 4.45-7.50), age≥80 years (aOR=3.15, 95%CI 2.26-4.40), arrhythmia (aOR=3.55, 95%CI 2.53-4.98), pulmonary hypertension (aOR=3.05, 95%CI 1.21-7.68), and prescription of anticoagulants (aOR=3.81, 95%CI 2.57-5.64), positive inotropes (aOR=2.29, 95%CI 1.41-3.74) and anti-arrhythmic drugs (aOR=2.14, 95%CI 1.10-4.15). CONCLUSIONS: Cardiopulmonary factors were associated with hospitalization for HF in the 30 days following a COPD exacerbation, while only cardiovascular-related factors and exacerbation severity were associated with AF hospitalization. Understanding factors will help target people for prevention.
Assuntos
Fibrilação Atrial , Flutter Atrial , Insuficiência Cardíaca , Hospitalização , Doença Pulmonar Obstrutiva Crônica , Humanos , Masculino , Doença Pulmonar Obstrutiva Crônica/epidemiologia , Doença Pulmonar Obstrutiva Crônica/complicações , Feminino , Estudos de Casos e Controles , Idoso , Fibrilação Atrial/epidemiologia , Insuficiência Cardíaca/epidemiologia , Flutter Atrial/epidemiologia , Pessoa de Meia-Idade , Fatores de Risco , Idoso de 80 Anos ou mais , Hospitalização/estatística & dados numéricos , Progressão da Doença , Modelos LogísticosRESUMO
The obligate intracellular pathogen Chlamydia trachomatis is a globally significant cause of sexually transmitted bacterial infections and the leading etiological agent of preventable blindness. The first-row transition metal iron (Fe) plays critical roles in chlamydial cell biology, and acquisition of this nutrient is essential for the survival and virulence of the pathogen. Nevertheless, how C. trachomatis acquires Fe from host cells is not well understood, since it lacks genes encoding known siderophore biosynthetic pathways, receptors for host Fe storage proteins, and the Fe acquisition machinery common to many bacteria. Recent studies have suggested that C. trachomatis directly acquires host Fe via the ATP-binding cassette permease YtgABCD. Here, we characterized YtgA, the periplasmic solute binding protein component of the transport pathway, which has been implicated in scavenging Fe(III) ions. The structure of Fe(III)-bound YtgA was determined at 2.0-Å resolution with the bound ion coordinated via a novel geometry (3 Ns, 2 Os [3N2O]). This unusual coordination suggested a highly plastic metal binding site in YtgA capable of interacting with other cations. Biochemical analyses showed that the metal binding site of YtgA was not restricted to interaction with only Fe(III) ions but could bind all transition metal ions examined. However, only Mn(II), Fe(II), and Ni(II) ions bound reversibly to YtgA, with Fe being the most abundant cellular transition metal in C. trachomatis Collectively, these findings show that YtgA is the metal-recruiting component of the YtgABCD permease and is most likely involved in the acquisition of Fe(II) and Mn(II) from host cells.IMPORTANCEChlamydia trachomatis is the most common bacterial sexually transmitted infection in developed countries, with an estimated global prevalence of 4.2% in the 15- to 49-year age group. Although infection is asymptomatic in more than 80% of infected women, about 10% of cases result in serious disease. Infection by C. trachomatis is dependent on the ability to acquire essential nutrients, such as the transition metal iron, from host cells. In this study, we show that iron is the most abundant transition metal in C. trachomatis and report the structural and biochemical properties of the iron-recruiting protein YtgA. Knowledge of the high-resolution structure of YtgA will provide a platform for future structure-based antimicrobial design approaches.
Assuntos
Antígenos de Bactérias/química , Proteínas de Ligação ao Ferro/química , Ferro/metabolismo , Antígenos de Bactérias/metabolismo , Sítios de Ligação , Proteínas de Ligação ao Ferro/metabolismoRESUMO
The opposing catalytic activities of topoisomerase I (TopoI/relaxase) and DNA gyrase (supercoiling enzyme) ensure homeostatic maintenance of bacterial chromosome supercoiling. Earlier studies in Escherichia coli suggested that the alteration in DNA supercoiling affects the DNA gyrase and TopoI expression. Although, the role of DNA elements around the promoters were proposed in regulation of gyrase, the molecular mechanism of supercoiling mediated control of TopoI expression is not yet understood. Here, we describe the regulation of TopoI expression from Mycobacterium tuberculosis and Mycobacterium smegmatis by a mechanism termed Supercoiling Sensitive Transcription (SST). In both the organisms, topoI promoter(s) exhibited reduced activity in response to chromosome relaxation suggesting that SST is intrinsic to topoI promoter(s). We elucidate the role of promoter architecture and high transcriptional activity of upstream genes in topoI regulation. Analysis of the promoter(s) revealed the presence of sub-optimal spacing between the -35 and -10 elements, rendering them supercoiling sensitive. Accordingly, upon chromosome relaxation, RNA polymerase occupancy was decreased on the topoI promoter region implicating the role of DNA topology in SST of topoI. We propose that negative supercoiling induced DNA twisting/writhing align the -35 and -10 elements to facilitate the optimal transcription of topoI.
Assuntos
DNA Girase/biossíntese , DNA Topoisomerases Tipo I/biossíntese , Homeostase/genética , Transcrição Gênica , DNA Girase/genética , DNA Topoisomerases Tipo I/genética , DNA Super-Helicoidal/genética , RNA Polimerases Dirigidas por DNA/genética , Regulação Bacteriana da Expressão Gênica , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Regiões Promotoras GenéticasRESUMO
The steady-state negative supercoiling of eubacterial genomes is maintained by the action of DNA topoisomerases. Topoisomerase distribution varies in different species of mycobacteria. While Mycobacterium tuberculosis (Mtb) contains a single type I (TopoI) and a single type II (Gyrase) enzyme, Mycobacterium smegmatis (Msm) and other members harbour additional relaxases. TopoI is essential for Mtb survival. However, the necessity of TopoI or other relaxases in Msm has not been investigated. To recognize the importance of TopoI for growth, physiology and gene expression of Msm, we have developed a conditional knock-down strain of TopoI in Msm. The TopoI-depleted strain exhibited extremely slow growth and drastic changes in phenotypic characteristics. The cessation of growth indicates the essential requirement of the enzyme for the organism in spite of having additional DNA relaxation enzymes in the cell. Notably, the imbalance in TopoI level led to the altered expression of topology modulatory proteins, resulting in a diffused nucleoid architecture. Proteomic and transcript analysis of the mutant indicated reduced expression of the genes involved in central metabolic pathways and core DNA transaction processes. RNA polymerase (RNAP) distribution on the transcription units was affected in the TopoI-depleted cells, suggesting global alteration in transcription. The study thus highlights the essential requirement of TopoI in the maintenance of cellular phenotype, growth characteristics and gene expression in mycobacteria. A decrease in TopoI level led to altered RNAP occupancy and impaired transcription elongation, causing severe downstream effects.
Assuntos
Proteínas de Bactérias/metabolismo , Núcleo Celular/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Núcleo Celular/química , Núcleo Celular/genética , DNA Topoisomerases Tipo I/genética , Regulação Bacteriana da Expressão Gênica , Mycobacterium smegmatis/química , Mycobacterium smegmatis/genética , FenótipoRESUMO
Bacterial DNA topoisomerase I (topoI) carries out relaxation of negatively supercoiled DNA through a series of orchestrated steps, DNA binding, cleavage, strand passage and religation. The N-terminal domain (NTD) of the type IA topoisomerases harbor DNA cleavage and religation activities, but the carboxyl terminal domain (CTD) is highly diverse. Most of these enzymes contain a varied number of Zn(2+) finger motifs in the CTD. The Zn(2+) finger motifs were found to be essential in Escherichia coli topoI but dispensable in the Thermotoga maritima enzyme. Although, the CTD of mycobacterial topoI lacks Zn(2+) fingers, it is indispensable for the DNA relaxation activity of the enzyme. The divergent CTD harbors three stretches of basic amino acids needed for the strand passage step of the reaction as demonstrated by a new assay. We also show that the basic amino acids constitute an independent DNA-binding site apart from the NTD and assist the simultaneous binding of two molecules of DNA to the enzyme, as required during the catalytic step. Although the NTD binds to DNA in a site-specific fashion to carry out DNA cleavage and religation, the basic residues in CTD bind to non-scissile DNA in a sequence-independent manner to promote the crucial strand passage step during DNA relaxation. The loss of Zn(2+) fingers from the mycobacterial topoI could be associated with Zn(2+) export and homeostasis.
Assuntos
Aminoácidos Básicos/metabolismo , Proteínas de Bactérias/metabolismo , DNA Topoisomerases Tipo I/metabolismo , DNA Bacteriano/genética , Mycobacterium smegmatis/enzimologia , Aminoácidos Básicos/genética , Proteínas de Bactérias/genética , Sítios de Ligação , Transporte Biológico , Clivagem do DNA , DNA Topoisomerases Tipo I/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutagênese Sítio-Dirigida , Mycobacterium smegmatis/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Dedos de ZincoRESUMO
BACKGROUND: Patient support programmes (PSPs) allow patients with chronic diseases to receive treatment and support at home. This study describes the Connect 360 PSP delivery and impact on patient-reported outcomes, satisfaction and adherence/persistence among benralizumab-treated patients with severe eosinophilic asthma (SEA). METHODS: A non-interventional retrospective cohort study using data collected during routine care in the Connect 360 PSP. All consenting enrollees (≥18 years) were included in the study. RESULTS: 746 patients formed the study cohort. Mean (SD) age was 53.7 (14.5) years on PSP entry; 38.3% were female (38.7% unknown). 79.6% of patients were experienced biological therapy users. Oral corticosteroid (OCS) use was reported in 48.4% of patients at baseline and 34.8% at 48 weeks. 8.2% of patients reported asthma hospitalisation in the previous 6 months at 24 weeks vs 3.0% at 48 weeks. Mean (SD) 6-item Asthma Control Questionnaire (ACQ-6) scores were 2.7 (1.5) at baseline vs 1.6 (1.3) at 48 weeks. Mean (SD) patient satisfaction scores remained high (4.5 of 5 (1.0) at baseline; 4.7 of 5 (0.6) at 48 weeks). 28.3% of patients were considered adherent at 24 weeks, increasing to 98.3% when supplemented with sales/delivery data (sensitivity analysis). Discontinuation from PSP/benralizumab was low at 24 (3.4%/3.0%) and 48 (12.6%/5.8%) weeks. CONCLUSIONS: Connect 360 PSP achieved high levels of satisfaction and persistence, with indications of positive outcomes including OCS use, hospitalisation and ACQ-6. The study was conducted during COVID-19, so it provides reassurance that patients with SEA receiving benralizumab may be supported safely and effectively at home.
Assuntos
Asma , Eosinofilia Pulmonar , Humanos , Feminino , Pessoa de Meia-Idade , Masculino , Estudos Retrospectivos , Anticorpos Monoclonais Humanizados , Reino UnidoRESUMO
BACKGROUND: Type 2 low-severe asthma phenotype is often a result of corticosteroid-overtreated type 2 disease owing to persistent symptoms, often unrelated to asthma and unlikely to respond to high-dose corticosteroid treatment. OBJECTIVE: This study aimed to characterize patients with severe asthma with low eosinophil counts (<300 cells/µL) and describe their disease burden and treatment across health care settings in the United Kingdom. METHODS: A retrospective cohort study of patients with severe asthma using linked Clinical Practice Research Datalink (CPRD) Aurum-Hospital Episode Statistics (HES) and UK Severe Asthma Registry (UKSAR) data indexed patients according to the latest blood eosinophil count (BEC). Clinical characteristics, treatment patterns, outcomes, and health care resource use were described by baseline BEC (≤150 and >150 to <300 cells/µL). RESULTS: Analysis included 701 (CPRD-HES) and 1,546 (UKSAR) patients; 60.5% and 59.4% had BECs 150 cells/µL or less at baseline, respectively. Across BEC groups, the proportion with uncontrolled asthma (two or more exacerbations) at follow-up (12 months after the index) was 5.4% in CPRD-HES and 45.2% in UKSAR. Maintenance oral corticosteroid use remained high across BEC groups (CPRD-HES: 29.4%; UKSAR: 51.7%), symptom control remained poor (>200 µg short-acting ß2 agonist or >500 µg terbutaline/d in CPRD-HES: 48.8%; median Asthma Control Questionnaire-6 score in UKSAR: 2.0 [range, 1.0-3.3]). Health care resource use was similar across BEC groups. CONCLUSIONS: Most patients managed in primary care experienced infrequent exacerbations, whereas UKSAR patients had frequent exacerbations. Large proportions of both patient groups had poor symptom control and continued to receive high levels of maintenance oral corticosteroids, increasing the risk of corticosteroid-induced morbidity. These data highlight the need for rigorous assessment of underlying disease pathology to guide appropriate treatment.
RESUMO
BACKGROUND: Chlamydia trachomatis infection results in reproductive damage in some women. The process and factors involved in this immunopathology are not well understood. This study aimed to investigate the role of primary human cellular responses to chlamydial stress response proteases and chlamydial infection to further identify the immune processes involved in serious disease sequelae. RESULTS: Laboratory cell cultures and primary human reproductive epithelial cultures produced IL-6 in response to chlamydial stress response proteases (CtHtrA and CtTsp), UV inactivated Chlamydia, and live Chlamydia. The magnitude of the IL-6 response varied considerably (up to 1000 pg ml(-1)) across different primary human reproductive cultures. Thus different levels of IL-6 production by reproductive epithelia may be a determinant in disease outcome. Interestingly, co-culture models with either THP-1 cells or autologous primary human PBMC generally resulted in increased levels of IL-6, except in the case of live Chlamydia where the level of IL-6 was decreased compared to the epithelial cell culture only, suggesting this pathway may be able to be modulated by live Chlamydia. PBMC responses to the stress response proteases (CtTsp and CtHtrA) did not significantly vary for the different participant cohorts. Therefore, these proteases may possess conserved innate PAMPs. MAP kinases appeared to be involved in this IL-6 induction from human cells. Finally, we also demonstrated that IL-6 was induced by these proteins and Chlamydia from mouse primary reproductive cell cultures (BALB/C mice) and mouse laboratory cell models. CONCLUSIONS: We have demonstrated that IL-6 may be a key factor for the chlamydial disease outcome in humans, given that primary human reproductive epithelial cell culture showed considerable variation in IL-6 response to Chlamydia or chlamydial proteins, and that the presence of live Chlamydia (but not UV killed) during co-culture resulted in a reduced IL-6 response suggesting this response may be moderated by the presence of the organism.
Assuntos
Proteínas de Bactérias/imunologia , Chlamydia trachomatis/imunologia , Células Epiteliais/imunologia , Interleucina-6/imunologia , Idoso , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Colo do Útero/citologia , Chlamydia trachomatis/fisiologia , Citocinas/imunologia , Citocinas/metabolismo , Endométrio/citologia , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Células HeLa , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interleucina-6/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-IdadeRESUMO
The genetic principle of synthetic lethality is clinically validated in cancers with loss of specific DNA damage response (DDR) pathway genes (i.e. BRCA1/2 tumor suppressor mutations). The broader question of whether and how oncogenes create tumor-specific vulnerabilities within DDR networks remains unanswered. Native FET protein family members are among the earliest proteins recruited to DNA double-strand breaks (DSBs) during the DDR, though the function of both native FET proteins and FET fusion oncoproteins in DSB repair remains poorly defined. Here we focus on Ewing sarcoma (ES), an EWS-FLI1 fusion oncoprotein-driven pediatric bone tumor, as a model for FET rearranged cancers. We discover that the EWS-FLI1 fusion oncoprotein is recruited to DNA DSBs and interferes with native EWS function in activating the DNA damage sensor ATM. Using preclinical mechanistic approaches and clinical datasets, we establish functional ATM deficiency as a principal DNA repair defect in ES and the compensatory ATR signaling axis as a collateral dependency and therapeutic target in FET rearranged cancers. Thus, aberrant recruitment of a fusion oncoprotein to sites of DNA damage can disrupt normal DSB repair, revealing a mechanism for how oncogenes can create cancer-specific synthetic lethality within DDR networks.
RESUMO
While oncogenes promote cancer cell growth, unrestrained proliferation represents a significant stressor to cellular homeostasis networks such as the DNA damage response (DDR). To enable oncogene tolerance, many cancers disable tumor suppressive DDR signaling through genetic loss of DDR pathways and downstream effectors (e.g., ATM or p53 tumor suppressor mutations). Whether and how oncogenes can help "self-tolerize" by creating analogous functional deficiencies in physiologic DDR networks is not known. Here we focus on Ewing sarcoma, a FET fusion oncoprotein (EWS-FLI1) driven pediatric bone tumor, as a model for the class of FET rearranged cancers. Native FET protein family members are among the earliest factors recruited to DNA double-strand breaks (DSBs) during the DDR, though the function of both native FET proteins and FET fusion oncoproteins in DNA repair remains to be defined. Using preclinical mechanistic studies of the DDR and clinical genomic datasets from patient tumors, we discover that the EWS-FLI1 fusion oncoprotein is recruited to DNA DSBs and interferes with native FET (EWS) protein function in activating the DNA damage sensor ATM. As a consequence of FET fusion-mediated interference with the DDR, we establish functional ATM deficiency as the principal DNA repair defect in Ewing sarcoma and the compensatory ATR signaling axis as a collateral dependency and therapeutic target in multiple FET rearranged cancers. More generally, we find that aberrant recruitment of a fusion oncoprotein to sites of DNA damage can disrupt physiologic DSB repair, revealing a mechanism for how growth-promoting oncogenes can also create a functional deficiency within tumor suppressive DDR networks.
RESUMO
Elevated levels of reactive oxygen species (ROS) reduce replication fork velocity by causing dissociation of the TIMELESS-TIPIN complex from the replisome. Here, we show that ROS generated by exposure of human cells to the ribonucleotide reductase inhibitor hydroxyurea (HU) promote replication fork reversal in a manner dependent on active transcription and formation of co-transcriptional RNA:DNA hybrids (R-loops). The frequency of R-loop-dependent fork stalling events is also increased after TIMELESS depletion or a partial inhibition of replicative DNA polymerases by aphidicolin, suggesting that this phenomenon is due to a global replication slowdown. In contrast, replication arrest caused by HU-induced depletion of deoxynucleotides does not induce fork reversal but, if allowed to persist, leads to extensive R-loop-independent DNA breakage during S-phase. Our work reveals a link between oxidative stress and transcription-replication interference that causes genomic alterations recurrently found in human cancer.
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA , Humanos , Espécies Reativas de Oxigênio , Fase S/genética , Proteínas de Ligação a DNA/metabolismo , Hidroxiureia/farmacologia , DNARESUMO
Background: This study compared management of high-risk COPD patients in the UK to national and international management recommendations and quality standards, including the COllaboratioN on QUality improvement initiative for achieving Excellence in STandards of COPD care (CONQUEST). The primary comparison was in 2019, but trends from 2000 to 2019 were also examined. Methods: Patients identified in the Optimum Patient Care Research Database were categorised as newly diagnosed (≤12 months after diagnosis), already diagnosed, and potential COPD (smokers having exacerbation-like events). High-risk patients had a history of ≥2 moderate or ≥1 severe exacerbations in the previous 12 months. Findings: For diagnosed patients, the median time between diagnosis and first meeting the high-risk criteria was 617 days (Q1-Q3: 3246). The use of spirometry for diagnosis increased dramatically after 2004 before plateauing and falling in recent years. In 2019, 41% (95% CI 39-44%; n = 550/1343) of newly diagnosed patients had no record of spirometry in the previous year, and 45% (95% CI 43-48%; n = 352/783) had no record of a COPD medication review within 6 months of treatment initiation or change. In 2019, 39% (n = 6893/17,858) of already diagnosed patients had no consideration of exacerbation rates, 46% (95% CI 45-47%; n = 4942/10,725) were not offered or referred for pulmonary rehabilitation, and 41% (95% CI 40-42%; n = 3026/7361) had not had a COPD review within 6 weeks of respiratory hospitalization. Interpretation: Opportunities for early diagnosis of COPD patients at high risk of exacerbations are being missed. Newly and already diagnosed patients at high-risk are not being assessed or treated promptly. There is substantial scope to improve the assessment and treatment optimisation of these patients. Funding: This study is conducted by the Observational & Pragmatic Research International Ltd and was co-funded by Optimum Patient Care and AstraZeneca. No funding was received by the Observational & Pragmatic Research Institute Pte Ltd (OPRI) for its contribution.
RESUMO
AIM: To assess the prevalence of metabolic syndrome and its correlation with the severity and duration of vitiligo. METHODS: One hundred vitiligo patients and 100 age-and-sex matched controls were included, whose waist circumference and blood pressure were measured; fasting serum cholesterol, triglycerides and glucose levels quantified; disease severity assessed and metabolic syndrome defined by National Cholesterol Education Program (NCEP) criteria. RESULTS: Metabolic syndrome (24%:12%), hypertriglyceridemia (41%:24%), impaired glucose tolerance (25%:16%) [P<0.05] and low HDL (P=0.044) were significantly more prevalent in cases as compared to controls as were the mean values of triglycerides and fasting blood sugar. Increased mean age of vitiligo patients correlated with the presence of metabolic syndrome. CONCLUSION: Metabolic syndrome had significant presence in but remained unaffected by the severity of vitiligo in our study patients.