RESUMO
PURPOSE: Here, we report the sensitivity of a personalized, tumor-informed circulating tumor DNA (ctDNA) assay (Signatera) for detection of molecular relapse during long-term follow-up of patients with breast cancer. METHODS: A total of 156 patients with primary breast cancer were monitored clinically for up to 12 years after surgery and adjuvant chemotherapy. Semiannual blood samples were prospectively collected, and analyzed retrospectively to detect residual disease by ultradeep sequencing using ctDNA assays, developed from primary tumor whole-exome sequencing data. RESULTS: Personalized Signatera assays detected ctDNA ahead of clinical or radiologic relapse in 30 of the 34 patients who relapsed (patient-level sensitivity of 88.2%). Relapse was predicted with a lead interval of up to 38 months (median, 10.5 months; range, 0-38 months), and ctDNA positivity was associated with shorter relapse-free survival (P < .0001) and overall survival (P < .0001). All relapsing triple-negative patients (n = 7/23) had a ctDNA-positive test within a median of 8 months (range, 0-19 months), while the 16 nonrelapsed patients with triple-negative breast cancer remained ctDNA-negative during a median follow-up of 58 months (range, 8-99 months). The four patients who had negative tests before relapse all had hormone receptor-positive (HR+) disease and conversely, five of the 122 nonrelapsed patients (all HR+) had an occasional positive test. CONCLUSION: Serial postoperative ctDNA assessment has strong prognostic value, provides a potential window for earlier therapeutic intervention, and may enable more effective monitoring than current clinical tests such as cancer antigen 15-3. Our study provides evidence that those with serially negative ctDNA tests have superior clinical outcomes, providing reassurance to patients with breast cancer. For select cases with HR+ disease, decisions about treatment management might require serial monitoring despite the ctDNA-positive result.
Assuntos
Neoplasias da Mama , DNA Tumoral Circulante , Humanos , Feminino , Neoplasias da Mama/genética , Neoplasias da Mama/sangue , Neoplasias da Mama/cirurgia , DNA Tumoral Circulante/sangue , Pessoa de Meia-Idade , Prognóstico , Seguimentos , Idoso , Adulto , Recidiva Local de Neoplasia/sangue , Recidiva Local de Neoplasia/genética , Estudos Retrospectivos , Idoso de 80 Anos ou maisRESUMO
PURPOSE: Although changes in circulating tumour DNA (ctDNA) in breast cancer are well described, the kinetics of their fluctuations has not been described over short timescales. We investigated ctDNA dynamics during alternating cycles of chemotherapy and hormonal treatment in pre-treated patients with oestrogen receptor-positive metastatic breast cancer. METHODS: Patients received alternating, 9-week cycles of eribulin and aromatase inhibitors (AIs). The clinical primary endpoint, progression-free survival (PFS), was monitored at 3, 6 and 9 months; secondary endpoints, clinical benefit rate (CBR), safety and tolerability profiles, were also assessed. Importantly, ctDNA fluctuations were monitored using the Oncomine™ Breast cfDNA assay to test whether biomarkers may change rapidly between chemotherapy and aromatase inhibitor (AI) treatment in the setting of advanced breast cancer, potentially reflecting disease dynamics. RESULTS: The median PFS was 202 days (95% CI: 135-undefined) and 235 days (95% CI: 235-undefined) at 6 and 9 months, respectively, with a 50% CBR at both 6 and 9 months. Dynamic changes in ctDNA were observed in short timescales between chemotherapy and AI treatment and support the clinical benefit (CB) seen in individual patients and, critically, appear informative of acquired resistance in real time. CONCLUSION: Changes in ctDNA can occur rapidly and reflect changes in patients' clinical tumour responses (NCT02681523).
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Inibidores da Aromatase , Neoplasias da Mama , DNA Tumoral Circulante , Adulto , Idoso , Feminino , Humanos , Pessoa de Meia-Idade , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Inibidores da Aromatase/uso terapêutico , Inibidores da Aromatase/administração & dosagem , Biomarcadores Tumorais/sangue , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/sangue , Neoplasias da Mama/mortalidade , Neoplasias da Mama/genética , DNA Tumoral Circulante/sangue , DNA Tumoral Circulante/genética , Furanos/uso terapêutico , Furanos/administração & dosagem , Cetonas , Metástase Neoplásica , Policetídeos de Poliéter , Resultado do TratamentoRESUMO
BACKGROUND: Programmed death ligand-1 (PD-L1) expression on circulating tumor cells (CTCs) has been suggested to provide prognostic information in non-small cell lung cancer (NSCLC), but consensus relative to treatment outcomes is lacking. We conducted the first comprehensive meta-analysis exploring its potential as a prognostic and predictive marker, and assessed the concordance between PD-L1 + CTCs and paired tumor tissue in NSCLC patients. METHOD: A comprehensive search was applied to PubMed and EMBASE to identify 26 studies that evaluated PD-L1 + CTCs and their association with survival outcomes in 1236 NSCLC patients. RESULTS: The meta-analysis estimated a mean PD-L1 + CTCs detection rate of 61% (95% CI, 49-72). Subgroup analysis based on treatment showed that PD-L1 + CTCs was not significantly associated with better overall survival (OS) in NSCLC patients treated with immune checkpoint inhibitors (ICIs) (Hazard Ratio (HR) = 0.96, 95% CI, 0.35-2.65, P = 0.944), but was predictive of worse OS in those treated with other therapies (HR = 2.11, 95% CI, 1.32-3.36, P = 0.002). Similarly, PD-L1 + CTCs was not significantly associated with superior progressing free survival (PFS) in NSCLCs treated with ICIs (HR = 0.67, 95% CI, 0.41-1.09, P = 0.121), but was significantly associated with shorter PFS in patients treated with other therapies (HR = 1.91, 95% CI, 1.24-2.94, P = 0.001). The overall estimate for the concordance between PD-L1 expression on CTCs and tumor cells was 63% (95% CI, 44-80). CONCLUSION: The average detection rate of PD-L1 + CTCs was comparable to the rate of PD-L1 expression in NSCLC tumors. There was a trend towards better PFS in ICI-treated NSCLC patients with PD-L1 + CTCs. Larger longitudinal studies on the association of PD-L1 + CTCs with clinical outcomes in NSCLC patients treated with ICIs are warranted.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Células Neoplásicas Circulantes , Humanos , Antígeno B7-H1 , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológicoRESUMO
BACKGROUND: Somatic copy number alterations (sCNAs) acquired during the evolution of breast cancer provide valuable prognostic and therapeutic information. Here we present a workflow for screening sCNAs using picogram amounts of cell-free DNA (cfDNA) and single circulating tumor cells (CTCs). METHODS: We repurposed the Ion ReproSeq PGS™ preimplantation genetic testing kit to perform shallow whole genome sequencing on 178 cfDNA samples (300 pg) and individual CTCs from 10 MBC patients with metastatic breast cancer (MBC) recovered by CellSearch®/DEPArray™. Results were analyzed using a tailored ichorCNA workflow. RESULTS: sCNAs were detected in cfDNA of 41/105 (39%) patients with MBC and 3/23 (13%) primary breast cancers on follow-up (PBC FU), all of whom subsequently relapsed. In 8 of 10 MBCs, individual CTCs had a higher copy number count than matched cfDNA. The median tumor fraction detected by ichorCNA was 0.34 (range 0.17-0.58) for MBC and 0.36 (range 0.31-0.37) for PBC FU. Patients with detectable tumor fraction (≥ 0.1) and TFx and OncomineTM variants had significantly lower overall survival rates (P values P = 0.002 and P < 0.0001 for the log-rank test, respectively). CONCLUSIONS: The ReproSeq PGS assay is rapid, at approximately $120 per sample, providing both a sCNA profile and estimation of the tumor DNA fraction from limiting cfDNA template (300pg) and individual CTCs. The approach could be used to examine the copy number landscape over time to guide treatment decisions, support future trial designs, and be applied to low volume blood spot samples enabling remote monitoring.
Assuntos
Neoplasias da Mama , Ácidos Nucleicos Livres , Células Neoplásicas Circulantes , Humanos , Feminino , Ácidos Nucleicos Livres/genética , Fluxo de Trabalho , Células Neoplásicas Circulantes/patologia , Neoplasias da Mama/patologia , Sequenciamento Completo do Genoma , Biomarcadores Tumorais/genéticaRESUMO
BACKGROUND: Little is known about how asymptomatic testing as a method to control transmission of COVID-19 can be implemented, and the prevalence of asymptomatic infection within university populations. The objective of this study was to investigate how to effectively set-up and implement a COVID-19 testing programme using novel reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) technology and to quantify the scale of asymptomatic infection on a university campus. METHODS: An observational study to describe the set-up and implementation of a novel COVID-19 testing programme on a UK university campus between September and December 2020. RT-LAMP testing was used to identify asymptomatic cases. RESULTS: A total of 1,673 tests were performed using RT-LAMP during the study period, of which 9 were positive for COVID-19, giving an overall positivity rate of 0.54%, equivalent to a rate in the tested population of 538 cases per 100,000 over the duration of testing. All positive tests were found to be positive on RT-PCR testing, giving a false positive rate of 0%. CONCLUSIONS: This study shows that it is possible to rapidly setup a universal university testing programme for COVID-19 in collaboration with local healthcare providers using RT-LAMP testing. Positive results were comparable to those in the local population, though with a different peak of infection. Further research to inform the design of the testing programme includes focus groups of those who underwent testing and further interrogation of the demographics of those opting to be tested to identify potential access problems or inequalities.
Assuntos
Teste para COVID-19 , COVID-19 , Humanos , COVID-19/diagnóstico , COVID-19/epidemiologia , SARS-CoV-2 , Infecções Assintomáticas , Sensibilidade e Especificidade , Técnicas de Diagnóstico Molecular/métodos , Reino Unido/epidemiologiaRESUMO
Mutations in the estrogen receptor (ESR1) gene are common in ER-positive breast cancer patients who progress on endocrine therapies. Most mutations localise to just three residues at, or near, the C-terminal helix 12 of the hormone binding domain, at leucine-536, tyrosine-537 and aspartate-538. To investigate these mutations, we have used CRISPR-Cas9 mediated genome engineering to generate a comprehensive set of isogenic mutant breast cancer cell lines. Our results confirm that L536R, Y537C, Y537N, Y537S and D538G mutations confer estrogen-independent growth in breast cancer cells. Growth assays show mutation-specific reductions in sensitivities to drugs representing three classes of clinical anti-estrogens. These differential mutation- and drug-selectivity profiles have implications for treatment choices following clinical emergence of ER mutations. Our results further suggest that mutant expression levels may be determinants of the degree of resistance to some anti-estrogens. Differential gene expression analysis demonstrates up-regulation of estrogen-responsive genes, as expected, but also reveals that enrichment for interferon-regulated gene expression is a common feature of all mutations. Finally, a new gene signature developed from the gene expression profiles in ER mutant cells predicts clinical response in breast cancer patients with ER mutations.
Assuntos
Neoplasias da Mama , Receptores de Estrogênio , Humanos , Feminino , Receptores de Estrogênio/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Prognóstico , Antagonistas de Estrogênios/uso terapêutico , Mutação , Estrogênios/farmacologiaRESUMO
BACKGROUND: We report copy-number profiling by low-pass WGS (LP-WGS) in individual circulating tumour cells (CTCs) for guiding treatment in patients with metastatic breast cancer (MBC), comparing CTC results with mutations detected in circulating tumour DNA (ctDNA) in the same blood samples. METHODS: Across 10 patients with MBC who were progressing at the time of blood sampling and that had >20 CTCs detected by CellSearch®, 63 single cells (50 CTCs and 13 WBCs) and 16 cell pools (8 CTC pools and 8 WBC pools) were recovered from peripheral blood by CellSearch®/DEPArray™ and sequenced with Ampli1 LowPass technology (Menarini Silicon Biosystems). Copy-number aberrations were identified using the MSBiosuite software platform, and results were compared with mutations detected in matched plasma cfDNA analysed by targeted next-generation sequencing using the Oncomine™ Breast cfDNA Assay (Thermo Fisher). RESULTS: LP-WGS data demonstrated copy-number gains/losses in individual CTCs in regions including FGFR1, JAK2 and CDK6 in five patients, ERBB2 amplification in two HER2-negative patients and BRCA loss in two patients. Seven of eight matched plasmas also had mutations in ctDNA in PIK3CA, TP53, ESR1 and KRAS genes with mutant allele frequencies (MAF) ranging from 0.05 to 33.11%. Combining results from paired CTCs and ctDNA, clinically actionable targets were identified in all ten patients. CONCLUSION: This combined analysis of CTCs and ctDNA may offer a new approach for monitoring of disease progression and to direct therapy in patients with advanced MBC, at a time when they are coming towards the end of other treatment options.
Assuntos
Neoplasias da Mama , Ácidos Nucleicos Livres , DNA Tumoral Circulante , Células Neoplásicas Circulantes , Humanos , Feminino , Neoplasias da Mama/patologia , Células Neoplásicas Circulantes/patologia , DNA Tumoral Circulante/genética , Ácidos Nucleicos Livres/genética , Mutação , Biomarcadores Tumorais/genéticaRESUMO
PURPOSE: To demonstrate the diagnostic performance of rapid SARS-CoV-2 RT-LAMP assays, comparing the performance of genomic versus sub-genomic sequence target with subsequent application in an asymptomatic screening population. METHODS: RT-LAMP diagnostic specificity (DSe) and sensitivity (DSe) was determined using 114 RT-PCR clinically positive and 88 RT-PCR clinically negative swab samples processed through the diagnostic RT-PCR service within the University Hospitals of Leicester NHS Trust. A swab-based RT-LAMP SARS-CoV-2 screening programme was subsequently made available to all staff and students at the University of Leicester (Autumn 2020), implemented to ISO 15189:2012 standards using NHS IT infrastructure and supported by University Hospital Leicester via confirmatory NHS diagnostic laboratory testing of RT-LAMP 'positive' samples. RESULTS: Validation samples reporting a Ct < 20 were detected at 100% DSe and DSp, reducing to 95% DSe (100% DSp) for all samples reporting a Ct < 30 (both genomic dual sub-genomic assays). Advisory screening identified nine positive cases in 1680 symptom free individuals (equivalent to 540 cases per 100,000) with results reported back to participants and feed into national statistics within 48 hours. CONCLUSION: This work demonstrates the utility of a rapid RT-LAMP assay for collapsing transmission of SARS-CoV-2 in an asymptomatic screening population.
Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , COVID-19/epidemiologia , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/genética , SARS-CoV-2/genética , Sensibilidade e EspecificidadeRESUMO
The mutagenic APOBEC3B (A3B) cytosine deaminase is frequently over-expressed in cancer and promotes tumour heterogeneity and therapy resistance. Hence, understanding the mechanisms that underlie A3B over-expression is important, especially for developing therapeutic approaches to reducing A3B levels, and consequently limiting cancer mutagenesis. We previously demonstrated that A3B is repressed by p53 and p53 mutation increases A3B expression. Here, we investigate A3B expression upon treatment with chemotherapeutic drugs that activate p53, including 5-fluorouracil, etoposide and cisplatin. Contrary to expectation, these drugs induced A3B expression and concomitant cellular cytosine deaminase activity. A3B induction was p53-independent, as chemotherapy drugs stimulated A3B expression in p53 mutant cells. These drugs commonly activate ATM, ATR and DNA-PKcs. Using specific inhibitors and gene knockdowns, we show that activation of DNA-PKcs and ATM by chemotherapeutic drugs promotes NF-κB activity, with consequent recruitment of NF-κB to the A3B gene promoter to drive A3B expression. Further, we find that A3B knockdown re-sensitises resistant cells to cisplatin, and A3B knockout enhances sensitivity to chemotherapy drugs. Our data highlight a role for A3B in resistance to chemotherapy and indicate that stimulation of A3B expression by activation of DNA repair and NF-κB pathways could promote cancer mutations and expedite chemoresistance.
Assuntos
Citidina Desaminase/genética , Antígenos de Histocompatibilidade Menor/genética , Neoplasias/genética , Fator de Transcrição RelA/genética , Proteína Supressora de Tumor p53/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Sistemas CRISPR-Cas/genética , Cisplatino/farmacologia , Reparo do DNA/efeitos dos fármacos , Etoposídeo/farmacologia , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Heterogeneidade Genética , Células HCT116 , Humanos , Células MCF-7 , Mutação/genética , NF-kappa B/genética , Neoplasias/patologiaRESUMO
P2X7 receptor (P2X7R) activation requires â¼100-fold higher concentrations of ATP than other P2X receptor (P2XR) subtypes. Such high levels are found during cellular stress, and P2X7Rs consequently contribute to a range of pathophysiological conditions. We have used chimeric and mutant P2X7Rs, coupled with molecular modeling, to produce a validated model of the binding mode of the subtype-selective antagonist A438079 at an intersubunit allosteric site. Within the allosteric site large effects on antagonist action were found for point mutants of residues F88A, D92A, F95A, and F103A that were conserved or similar between sensitive/insensitive P2XR subtypes, suggesting that these side-chain interactions were not solely responsible for high-affinity antagonist binding. Antagonist sensitivity was increased with mutations that remove the bulk of side chains around the center of the binding pocket, suggesting that the dimensions of the pocket make a significant contribution to selectivity. Chimeric receptors swapping the left flipper (around the orthosteric site) reduced both ATP and antagonist sensitivity. Point mutations within this region highlighted the contribution of a P2X7R-specific aspartic acid residue (D280) that modeling suggests forms a salt bridge with the lower body region of the receptor. The D280A mutant removing this charge increased ATP potency 15-fold providing a new insight into the low ATP sensitivity of the P2X7R. The ortho- and allosteric binding sites form either side of the ß-strand Y291-E301 adjacent to the left flipper. This structural linking may explain the contribution of the left flipper to both agonist and antagonist action.
Assuntos
Acetamidas/farmacologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Piridinas/farmacologia , Quinolinas/farmacologia , Receptores Purinérgicos P2X7/efeitos dos fármacos , Tetrazóis/farmacologia , Regulação Alostérica , Sítio Alostérico , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Ligantes , Simulação de Acoplamento Molecular , Mutação Puntual , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/genética , Homologia de Sequência de AminoácidosRESUMO
The P2X7 receptor (P2X7R) for ATP is a therapeutic target for pathophysiological states including inflammation, pain management and epilepsy. This is facilitated by the predicted low side effect profile as the high concentrations of ATP required to activate the receptor are usually only found following cell damage/disease and so P2X7Rs respond to a "danger" signal and are not normally active. AZ10606120 is a selective antagonist for P2X7Rs (IC50 of ~10 nM) and ineffective at the P2X1R (at 10 µM). To determine the molecular basis of selectivity we generated a series of P2X7/1R chimeras and mutants. Two regions that are unique to the P2X7R, a loop insertion (residues 73-79) and threonine residues T90 and T94, are required for high affinity antagonist action. Point mutations ruled out an orthosteric antagonist site. Mutations and molecular modelling identified an allosteric binding site that forms at the subunit interface at the apex of the receptor. Molecular dynamics simulations indicated that unique P2X7R features regulate access of AZ10606120 to the allosteric site. The characterisation of the allosteric pocket provides a new and novel target for rational P2X7R drug development.
Assuntos
Adamantano/análogos & derivados , Trifosfato de Adenosina/metabolismo , Sítio Alostérico , Aminoquinolinas/química , Sítios de Ligação , Ativação do Canal Iônico , Antagonistas do Receptor Purinérgico P2X/química , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/metabolismo , Adamantano/química , Adamantano/farmacologia , Alelos , Regulação Alostérica , Sequência de Aminoácidos , Substituição de Aminoácidos , Aminoquinolinas/farmacologia , Humanos , Lignanas , Modelos Moleculares , Conformação Molecular , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X7/genética , Relação Estrutura-AtividadeRESUMO
P2X7 receptors are ATP-gated ion channels that contribute to inflammation and cell death. They have the novel property of showing marked facilitation to repeated applications of agonist, and the intrinsic channel pore dilates to allow the passage of fluorescent dyes. A 60-s application of ATP to hP2X7 receptors expressed in Xenopus oocytes gave rise to a current that had a biphasic time course with initial and secondary slowly developing components. A second application of ATP evoked a response with a more rapid time to peak. This facilitation was reversed to initial levels following a 10-min agonist-free interval. A chimeric approach showed that replacement of the pre-TM1 amino-terminal region with the corresponding P2X2 receptor section (P2X7-2Nß) gave responses that quickly reached a steady state and did not show facilitation. Subsequent point mutations of variant residues identified Asn-16 and Ser-23 as important contributors to the time course/facilitation. The P2X7 receptor is unique in having an intracellular carboxyl-terminal cysteine-rich region (Ccys). Deletion of this region removed the secondary slowly developing current, and, when expressed in HEK293 cells, ethidium bromide uptake was only â¼5% that of WT levels, indicating reduced large pore formation. Dye uptake was also reduced for the P2X7-2Nß chimera. Surprisingly, combination of the chimera and the Ccys deletion (P2X7-2NßdelCcys) restored the current rise time and ethidium uptake to WT levels. These findings suggest that there is a coevolved interaction between the juxtatransmembrane amino and carboxyl termini in the regulation of P2X7 receptor gating.
Assuntos
Trifosfato de Adenosina/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Animais , Células HEK293 , Humanos , Mutagênese , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/genética , Xenopus laevisRESUMO
P2X receptor subtypes can be distinguished by their sensitivity to ATP analogues and selective antagonists. We have used chimeras between human P2X1 and P2X2 receptors to address the contribution of the extracellular ligand binding loop, transmembrane segments (TM1 and TM2), and intracellular amino and carboxyl termini to the action of partial agonists (higher potency and efficacy of BzATP and Ap5A at P2X1 receptors) and antagonists. Sensitivity to the antagonists NF449, suramin, and PPADS was conferred by the nature of the extracellular loop (e.g. nanomolar for NF449 at P2X1 and P2X2-1EXT and micromolar at P2X2 and P2X1-2EXT). In contrast, the effectiveness of partial agonists was similar to P2X1 levels for both of the loop transfers, suggesting that interactions with the rest of the receptor played an important role. Swapping TM2 had reciprocal effects on partial agonist efficacy. However, TM1 swaps increased partial agonist efficacy at both chimeras, and this was similar for swaps of both TM1 and 2. Changing the amino terminus had no effect on agonist potency but increased partial agonist efficacy at P2X2-1N and decreased it at P2X1-2N chimeras, demonstrating that potency and efficacy can be independently regulated. Chimeras and point mutations also identified residues in the carboxyl terminus that regulated recovery from channel desensitization. These results show that interactions among the intracellular, transmembrane, and extracellular portions of the receptor regulate channel properties and suggest that transitions to channel opening, the behavior of the open channel, and recovery from the desensitized state can be controlled independently.
Assuntos
Ativação do Canal Iônico/efeitos dos fármacos , Agonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X1/química , Receptores Purinérgicos P2X1/metabolismo , Receptores Purinérgicos P2X2/química , Receptores Purinérgicos P2X2/metabolismo , Proteínas Recombinantes/química , Trifosfato de Adenosina/farmacologia , Animais , Espaço Extracelular/química , Humanos , Espaço Intracelular/química , Modelos Biológicos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Antagonistas do Receptor Purinérgico P2X , Fosfato de Piridoxal/farmacologia , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Fatores de Tempo , Xenopus laevisRESUMO
P2X receptors for ATP have a wide range of physiological roles and comprise a structurally distinct family of ligand-gated trimeric ion channels. The crystal structure of a P2X4 receptor, in combination with mutagenesis studies, has provided a model of the intersubunit ATP-binding sites and identified an extracellular lateral portal, adjacent to the membrane, that funnels ions to the channel pore. However, little is known about the extent of ATP-induced conformational changes in the extracellular domain of the receptor. To address this issue, we have used MTSEA-biotinylation (N-Biotinoylaminoethyl methanethiosulfonate) to show ATP-sensitive accessibility of cysteine mutants at the human P2X1 receptor. Mapping these data to a P2X1 receptor homology model identifies significant conformational rearrangement. Electron microscopy of purified P2X1 receptors showed marked changes in structure on ATP binding, and introducing disulphide bonds between adjacent subunits to restrict intersubunit movements inhibited channel function. These results are consistent with agonist-induced rotation of the propeller-head domain of the receptor, sliding of adjacent subunits leading to restricted access to the upper vestibule, movement in the ion conducting lateral portals, and gating of the channel pore.
Assuntos
Receptores Purinérgicos P2X1/química , Trifosfato de Adenosina/química , Animais , Sítios de Ligação , Biotinilação , Dissulfetos/química , Humanos , Íons/química , Microscopia Eletrônica/métodos , Conformação Molecular , Mutagênese , Oócitos/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , XenopusRESUMO
P2X receptors show marked variations in the time-course of response to ATP application from rapidly desensitizing P2X1 receptors to relatively sustained P2X2 receptors. In this study we have used chimeras between human P2X1 and P2X2 receptors in combination with mutagenesis to address the contribution of the extracellular ligand binding loop, the transmembrane channel, and the intracellular regions to receptor time-course. Swapping either the extracellular loop or both transmembrane domains (TM1 and -2) between the P2X1 and P2X2 receptors had no effect on the time-course of ATP currents in the recipient receptor. These results suggest that the agonist binding and channel-forming portions of the receptor do not play a major role in the control of the time-course. In contrast replacing the amino terminus of the P2X1 receptor with that from the non-desensitizing P2X2 receptor (P2X1-2N) slowed desensitization, and the mirror chimera induced rapid desensitization in the P2X2-1N chimera. These reciprocal effects on time-course can be replicated by changing four variant amino acids just before the first transmembrane (TM1) segment. These pre-TM1 residues also had a dominant effect on chimeras where both TMs had been transferred; mutating the variant amino acids 21-23 to those found in the P2X2 receptor removed desensitization from the P2X1-2TM1/-2 chimera, and the reciprocal mutants induced rapid desensitization in the non-desensitizing P2X2-1TM1/-2 chimera. These results suggest that the intracellular amino terminus, in particular the region just before TM1, plays a dominant role in the regulation of the time-course of ATP evoked P2X receptor currents.
Assuntos
Receptores Purinérgicos P2X1/metabolismo , Receptores Purinérgicos P2X2/metabolismo , Animais , Humanos , Transporte de Íons/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores Purinérgicos P2X1/genética , Receptores Purinérgicos P2X2/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Xenopus laevisRESUMO
P2X receptors are ATP-gated cation channels. The x-ray structure of a P2X4 receptor provided a major advance in understanding the molecular basis of receptor properties. However, how agonists are coordinated, the extent of the binding site, and the contribution of the vestibules in the extracellular domain to ionic permeation have not been addressed. We have used cysteine-scanning mutagenesis to determine the contribution of residues Glu(52)-Gly(96) to human P2X1 receptor properties. ATP potency was reduced for the mutants K68C, K70C, and F92C. The efficacy of the partial agonist BzATP was also reduced for several mutants forming the back of the proposed agonist binding site. Molecular docking in silico of both ATP and BzATP provided models of the agonist binding site consistent with these data. Individual cysteine mutants had no effect or slightly increased antagonism by suramin or pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate. Mutants at the entrance to and lining the upper vestibule were unaffected by cysteine-reactive methanethiosulfonate (MTS) reagents, suggesting that it does not contribute to ionic permeation. Mutants that were sensitive to modification by MTS reagents were predominantly found either around the proposed ATP binding pocket or on the strands connecting the binding pocket to the transmembrane region and lining the central vestibule. In particular, ATP sensitivity and currents were increased by a positively charged MTS reagent at the G60C mutant at the interface between the central and extracellular vestibule. This suggests that dilation of the base of the central vestibule contributes to gating of the receptor.
Assuntos
Trifosfato de Adenosina/metabolismo , Ativação do Canal Iônico/fisiologia , Modelos Moleculares , Mutagênese , Receptores Purinérgicos P2X1/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Animais , Sítios de Ligação/fisiologia , Humanos , Mutação de Sentido Incorreto , Mapeamento de Peptídeos , Receptores Purinérgicos P2X1/química , Receptores Purinérgicos P2X1/genética , Xenopus laevisRESUMO
Cholesterol-rich lipid rafts act as signaling microdomains and can regulate receptor function. We have shown in HEK293 cells recombinant P2X1-4 receptors (ATP-gated ion channels) are expressed in lipid rafts. Localization to flotillin-rich lipid rafts was reduced by the detergent Triton X-100. This sensitivity to Triton X-100 was concentration- and subunit-dependent, demonstrating differential association of P2X1-4 receptors with lipid rafts. The importance of raft association to ATP-evoked P2X receptor responses was determined in patch clamp studies. The cholesterol-depleting agents methyl-ß-cyclodextrin or filipin disrupt lipid rafts and reduced P2X1 receptor currents by >90%. In contrast, ATP-evoked P2X2-4 receptor currents were unaffected by lipid raft disruption. To determine the molecular basis of cholesterol sensitivity, we generated chimeric receptors replacing portions of the cholesterol-sensitive P2X1 receptor with the corresponding region from the insensitive P2X2 receptor. These chimeras identified the importance of the intracellular amino-terminal region between the conserved protein kinase C site and the first transmembrane segment for the sensitivity to cholesterol depletion. Mutation of any of the variant residues between P2X1 and P2X2 receptors in this region in the P2X1 receptor (residues 20-23 and 27-29) to cysteine removed cholesterol sensitivity. Cholesterol depletion did not change the ATP sensitivity or cell surface expression of P2X1 receptors. This suggests that cholesterol is normally needed to facilitate the opening/gating of ATP-bound P2X1 receptor channels, and mutations in the pre-first transmembrane segment region remove this requirement.
Assuntos
Colesterol/metabolismo , Microdomínios da Membrana/metabolismo , Receptores Purinérgicos P2X1/metabolismo , Receptores Purinérgicos P2X2/metabolismo , Receptores Purinérgicos P2X3/metabolismo , Receptores Purinérgicos P2X4/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Linhagem Celular , Colesterol/genética , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Microdomínios da Membrana/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Mutação , Agonistas do Receptor Purinérgico P2X , Receptores Purinérgicos P2X1/agonistas , Receptores Purinérgicos P2X1/genética , Receptores Purinérgicos P2X2/genética , Receptores Purinérgicos P2X3/genética , Receptores Purinérgicos P2X4/genética , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
P2X1 receptors for ATP contribute to signalling in a variety of cell types and following stimulation undergo rapid desensitisation (within 1 s), and require approximately 5 min to recover. In HEK293 cells P2X1 receptors C-terminally tagged with enhanced green fluorescent protein (P2X1-eGFP) were predominantly expressed at the cell surface. Following > 90% photo-bleaching of P2X1-eGFP within a 6 microm(2) circle at the cell surface fluorescence recovery after photo-bleaching (FRAP) was fit with a time constant of approximately 60 s and recovered to approximately 75% of pre-bleach levels. Following activation of the P2X1 receptor with alpha,beta-methylene ATP the associated calcium influx doubled the FRAP recovery rate. The protein synthesis inhibitor cycloheximide had only a small effect on repeated FRAP and indicated a limited contribution of new P2X1 receptors to the FRAP. Inhibition of trafficking with brefeldin A reduced recovery and this effect could be reversed following receptor activation. In contrast, the dynamin inhibitor dynasore had no effect on FRAP under unstimulated conditions but reduced the level of recovery following agonist stimulation. In functional studies both brefeldin A and dynasore increased the recovery time from desensitisation. Taken together these studies demonstrate for the first time an important role of receptor recycling on P2X1 receptor responsiveness.
Assuntos
Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Brefeldina A/farmacologia , Linhagem Celular , Cicloeximida/farmacologia , Interpretação Estatística de Dados , Dinaminas/antagonistas & inibidores , Eletrofisiologia , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde , Humanos , Hidrazonas/farmacologia , Microscopia Confocal , Mutagênese Insercional , Técnicas de Patch-Clamp , Inibidores da Síntese de Proteínas/farmacologia , Agonistas do Receptor Purinérgico P2 , Receptores Purinérgicos P2X , Sistemas do Segundo Mensageiro/efeitos dos fármacosRESUMO
At the majority of mutants in the region Glu181-Val200 incorporating a conserved AsnPheThrPhiPhixLys motif cysteine substitution had no effect on sensitivity to ATP, partial agonists, or methanethiosulfonate (MTS) compounds. For the F185C mutant the efficacy of partial agonists was reduced by approximately 90% but there was no effect on ATP potency or the actions of MTS reagents. At T186C, F188C and K190C mutants ATP potency and partial agonists responses were reduced. The ATP sensitivity of the K190C mutant was rescued towards WT levels by positively charged (2-aminoethyl)methanethiosulfonate hydrobromide and reduced by negatively charged sodium (2-sulfonatoethyl) methanethiosulfonate. Both MTS reagents decreased ATP potency at the T186C mutant, and abolished responses at the F195C mutant. (32)P-2-azido ATP binding to the mutants T186C and K190C was sensitive to MTS reagents consistent with an effect on binding, however binding at F195C was unaffected indicating an effect on gating. The accessibility of the introduced cysteines was probed with (2-aminoethyl)methanethiosulfonate hydrobromide-biotin, this showed that the region Thr186-Ser192 is likely to form a beta sheet and that accessibility is blocked by ATP. Taken together these results suggest that Thr186, Phe188 and Lys190 are involved in ATP binding to the receptor and Phe185 and Phe195 contribute to agonist evoked conformational changes.