RESUMO
Nanopore sensors are a highly attractive platform for single-molecule sensing for sequencing, disease diagnostics, and drug screening. Outer membrane protein G (OmpG) nanopores have advantages for single-molecule sensing owing to their rigid monomeric structure, which comprises seven flexible loops, providing distinct gating patterns upon analyte binding. Blocking of the protein-protein interaction between B-cell lymphoma-extra-large (Bcl-xL) and the BH3 domain of Bcl-2 homologous antagonist/killer (Bak-BH3) has been reported as a promising strategy for anticancer therapy. Here, we characterized the interaction between Bcl-xL and Bak-BH3 as well as its inhibition by a small-molecule inhibitor using click chemistry-based Bak-BH3 peptide-conjugated OmpG nanopores. The binding of Bcl-xL to Bak-BH3 generated characteristic gating signals involving significant changes in the amplitudes of noise and gating parameters such as gating frequency, open probability, and durations of open and closed states. Notably, specific inhibition of Bcl-xL by the small-molecule antagonist, ABT-737, led to the recovery of the noise and gating parameters. Collectively, these results revealed that the chemically modified OmpG nanopore can serve as a valuable sensor platform for ultrasensitive, rapid, and single-molecule-based drug screening against protein-protein interactions, which are therapeutic targets for various diseases.
Assuntos
Proteínas de Escherichia coli , Nanoporos , Apoptose , Proteínas da Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/metabolismo , Nanotecnologia , Porinas/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMO
Apoptosis plays an essential role in maintaining cellular homeostasis and preventing cancer progression. Bcl-xL, an anti-apoptotic protein, is an important modulator of the mitochondrial apoptosis pathway and is a promising target for anticancer therapy. In this study, we identified octenidine as a novel Bcl-xL inhibitor through structural feature-based deep learning and molecular docking from a library of approved drugs. The NMR experiments demonstrated that octenidine binds to the Bcl-2 homology 3 (BH3) domain-binding hydrophobic region that consists of the BH1, BH2, and BH3 domains in Bcl-xL. A structural model of the Bcl-xL/octenidine complex revealed that octenidine binds to Bcl-xL in a similar manner to that of the well-known Bcl-2 family protein antagonist ABT-737. Using the NanoBiT protein-protein interaction system, we confirmed that the interaction between Bcl-xL and Bak-BH3 domains within cells was inhibited by octenidine. Furthermore, octenidine inhibited the proliferation of MCF-7 breast and H1299 lung cancer cells by promoting apoptosis. Taken together, our results shed light on a novel mechanism in which octenidine directly targets anti-apoptotic Bcl-xL to trigger mitochondrial apoptosis in cancer cells.
Assuntos
Inteligência Artificial , Iminas/farmacologia , Piridinas/farmacologia , Proteína bcl-X/antagonistas & inibidores , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Iminas/química , Simulação de Acoplamento Molecular , Neoplasias/patologia , Ligação Proteica/efeitos dos fármacos , Piridinas/química , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína bcl-X/químicaRESUMO
Bacterial riboswitch RNAs are attractive targets for novel antibiotics against antibiotic-resistant superbacteria. Their binding to cognate metabolites is essential for the regulation of bacterial gene expression. Despite the importance of RNAs as therapeutic targets, the development of RNA-targeted, small-molecule drugs is limited by current biophysical methods. Here, we monitored the specific interaction between the adenine-sensing riboswitch aptamer domain (ARS) and adenine at the single-molecule level using α-hemolysin (αHL) nanopores. During adenine-induced tertiary folding, adenine-bound ARS intermediates exhibited characteristic nanopore events, including a two-level ionic current blockade and a â¼ 5.6-fold longer dwell time than that of free RNA. In a proof-of-concept experiment, tertiary RNA folding-targeted drug screening was performed using a protein nanopore, which resulted in the discovery of three new ARS-targeting hit compounds from a natural compound library. Taken together, these results reveal that αHL nanopores are a valuable platform for ultrasensitive, label-free, and single-molecule-based drug screening against therapeutic RNA targets.
Assuntos
Nanoporos , Riboswitch , Avaliação Pré-Clínica de Medicamentos , Proteínas Hemolisinas , Dobramento de RNARESUMO
Signal transducer and activator of transcription 3 (STAT3) is aberrantly activated in many human cancers. We tried to find STAT3 inhibitors from natural sources and found that Xanthium fruit extracts decreased phosphorylation of STAT3-Y705. 8-Epi-xanthatin (EXT) was isolated from the extracts. When DU145 cancer cells were treated with EXT, p-STAT3-Y705 was decreased with an IC50 of 3.2 µM. EXT decreased the expression of STAT3 target genes, such as cyclin A, cyclin D1, and BCL-2, and induced PARP cleavage, indicating apoptotic cell death. Downregulation of EXT-induced p-STAT3-Y705 was rescued by pretreating DU145 cells with antioxidants, such as N-acetyl-L-cysteine (NAC), indicating that reactive oxygen species (ROS) were involved in the EXT-induced inhibition of STAT3 activation. Furthermore, we proved the association of EXT with STAT3 protein by using a drug affinity responsive target stability (DARTS) assay and a cellular thermal shift assay (CETSA). EXT inhibited proliferation of DU145 cells with a GI50 of 6 µM and reduced tumor growth in mice xenografted with DU145 cells. Immunoblotting showed that phosphorylation of STAT3-Y705 was lower in EXT-treated tumor tissue than in control tissues. Collectively, we found that EXT binds to, and inhibits, STAT3 activation and could be a lead compound for anticancer therapy.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Frutas/química , Furanos/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Fator de Transcrição STAT3/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Furanos/farmacologia , Humanos , Masculino , Camundongos , Camundongos Nus , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Neuraminidase (NA), one of the major surface glycoproteins of influenza A virus (IAV), is an important diagnostic biomarker and antiviral therapeutic target. Cytolysin A (ClyA) is a nanopore sensor with an internal constriction of 3.3 nm, enabling the detection of protein conformations at the single-molecule level. In this study, a nanopore-based approach is developed for analysis of the enzymatic activity of NA, which facilitates rapid and highly sensitive diagnosis of IAV. Current blockade analysis of the d-glucose/d-galactose-binding protein (GBP) trapped within a type I ClyA-AS (ClyA mutant) nanopore reveals that galactose cleaved from sialyl-galactose by NA of the influenza virus can be detected in real time and at the single-molecule level. Our results show that this nanopore sensor can quantitatively measure the activity of NA with 40-80-fold higher sensitivity than those previously reported. Furthermore, the inhibition of NA is monitored using small-molecule antiviral drugs, such as zanamivir. Taken together, our results reveal that the ClyA protein nanopore can be a valuable platform for the rapid and sensitive point-of-care diagnosis of influenza and for drug screening against the NA target.
Assuntos
Citotoxinas/metabolismo , Ensaios Enzimáticos/métodos , Vírus da Influenza A/enzimologia , Nanoporos , Neuraminidase/metabolismo , Citotoxinas/química , Modelos Moleculares , Neuraminidase/química , Conformação ProteicaRESUMO
In response to genotoxic stress, the tumor suppressor protein p73 induces apoptosis and cell cycle arrest. Despite extensive studies on p73-mediated apoptosis, little is known about the cytoplasmic apoptotic function of p73. Here, using H1299 lung cancer cells and diverse biochemical approaches, including colony formation, DNA fragmentation, GST pulldown, and apoptosis assays along with NMR spectroscopy, we show that p73 induces transcription-independent apoptosis via its transactivation domain (TAD) through a mitochondrial pathway and that this apoptosis is mediated by the interaction between p73-TAD and the anti-apoptotic protein B-cell lymphoma-extra large (Bcl-XL or BCL2L1). This binding disrupted an interaction between Bcl-XL and the pro-apoptotic protein BH3-interacting domain death agonist (Bid). In particular, we found that a 16-mer p73-TAD peptide motif (p73-TAD16) mediates transcription-independent apoptosis, accompanied by cytochrome c release from the mitochondria, by interacting with Bcl-XL Interestingly, the structure of the Bcl-XL-p73-TAD16 peptide complex revealed a novel mechanism of Bcl-XL recognition by p73-TAD. We observed that the α-helical p73-TAD16 peptide binds to a noncanonical site in Bcl-XL, comprising the BH1, BH2, and BH3 domains in an orientation opposite to those of pro-apoptotic BH3 peptides. Taken together, our results indicate that the cytoplasmic apoptotic function of p73 is mediated through a noncanonical mode of Bcl-XL recognition. This finding sheds light on a critical transcription-independent, p73-mediated mechanism for apoptosis induction, which has potential implications for anticancer therapy.
Assuntos
Apoptose , Citoplasma/metabolismo , Proteína Tumoral p73/metabolismo , Proteína bcl-X/metabolismo , Linhagem Celular Tumoral , Citoplasma/patologia , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Transcrição Gênica , Proteína Tumoral p73/química , Proteína bcl-X/genéticaRESUMO
Mitochondrial E3 ubiquitin ligase 1 (MUL1) is a multifunctional mitochondrial protein involved in various biological processes such as mitochondrial dynamics, cell growth, apoptosis, and mitophagy. MUL1 mediates the ubiquitylation of mitochondrial p53 for proteasomal degradation. Although the interaction of MUL1-RING domain with its substrate, p53, is a unique mechanism in RING-mediated ubiquitylation, the molecular basis of this process remains unknown. In this study, we determined the solution structure of the MUL1-RING domain and characterized its interaction with the p53 transactivation domain (p53-TAD) by nuclear magnetic resonance (NMR) spectroscopy. The overall structure of the MUL1-RING domain is similar to those of RING domains of other E3 ubiquitinases. The MUL1-RING domain adopts a ßßαß fold with three anti-parallel ß-strands and one α-helix, containing a canonical cross-brace motif for the ligation of two zinc ions. Through NMR chemical shift perturbation experiments, we determined the p53-TAD-binding site in the MUL1-RING domain and showed that the MUL1-RING domain interacts mainly with the p53-TAD2 subdomain composed of residues 39-57. Taken together, our results provide a molecular basis for the novel recognition mechanism of the p53-TAD substrate by the MUL1-RING domain.
Assuntos
Espectroscopia de Ressonância Magnética , Domínios RING Finger , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Humanos , Ligação Proteica , Especificidade por Substrato , UbiquitinaçãoRESUMO
Irinotecan is a strong anticancer drug whose mechanism of action has been reported only for the inhibition of DNA topoisomerase I (Topo I) through its active metabolite SN-38. In this study, we present a new mechanism of Irinotecan which inhibits the activities of MDM2, an E3 ligase of tumour suppressor p53, and Bcl-xL, an anti-apoptotic protein, through direct binding. In our structure modelling study, Irinotecan could fit to the binding sites of MDM2 and Bcl-xL for their known drugs, Nutlin-3 and ABT-737, with a better binding affinity than to Topo I. The direct binding of Irinotecan to both proteins was confirmed through a NMR study. We further showed that Irinotecan increased the amount of p53 only in the presence of MDM2 and inhibited the physical interaction of Bcl-xL with Bim, a core pro-apoptotic protein. In addition, we demonstrated that Irinotecan induced the down regulation of proliferation and strong G2/M arrest in HCT116 colon cancer cells shortly after treatment. Collectively, we suggest a new mechanism of action for Irinotecan as a dual target inhibitor of MDM2 and Bcl-xL facilitating the anticancer activities mediated by p53 and Bcl-xL interaction partners.
Assuntos
Irinotecano/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/metabolismo , Apoptose/efeitos dos fármacos , Proteína 11 Semelhante a Bcl-2/metabolismo , Sítios de Ligação , Compostos de Bifenilo/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/metabolismo , Células HCT116 , Humanos , Imidazóis/farmacologia , Irinotecano/química , Modelos Moleculares , Nitrofenóis/farmacologia , Ressonância Magnética Nuclear Biomolecular , Piperazinas/farmacologia , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/química , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Proteína bcl-X/químicaRESUMO
DJ-1 is a multifunctional protein associated with Parkinson's disease (PD) and tumorigenesis. In response to ultraviolet B (UVB) irradiation, DJ-1 is translocated into the mitochondria, and its interaction with the mitochondrial protein Bcl-XL protects cells against death. In this study, we characterized the molecular interaction between DJ-1 and Bcl-XL by NMR spectroscopy. The NMR chemical shift perturbation data demonstrated that the oxidized but not the reduced form of DJ-1 binds to the predominantly hydrophobic groove surrounded by the BH1-BH3 domains in Bcl-XL. In addition, our results showed that the C-terminal α8-helix peptide (Cpep) of DJ-1 binds to the pro-apoptotic BH3 peptide-binding hydrophobic groove in Bcl-XL and, thus, acts as a Bcl-XL-binding motif. In combination with the NMR chemical shift perturbation data, a refined structural model of the Bcl-XL/DJ-1 Cpep complex revealed that the binding mode is remarkably similar to that of other Bcl-XL/pro-apoptotic BH3 peptide complexes. Taken together, our results provide a structural basis for the binding mechanism between DJ-1 and Bcl-XL, which will contribute to molecular understanding of the role of mitochondrial DJ-1 in Bcl-XL regulation in response to oxidative stress.
Assuntos
Simulação de Acoplamento Molecular/métodos , Proteína Desglicase DJ-1/química , Mapeamento de Interação de Proteínas/métodos , Proteína bcl-X/química , Proteína bcl-X/ultraestrutura , Sítios de Ligação , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética/métodos , Modelos Químicos , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Relação Estrutura-AtividadeRESUMO
Eukaryotic transcription initiation is mediated by interactions between transcriptional activators and the mediator coactivator complex. Molecular interaction of p53 transcription factor with mediator complex subunit 25 (MED25) is essential for its target gene transcription. In this study, we characterized the molecular interaction between p53 transactivation domain (p53TAD) and activator interaction domain (ACID) of MED25 using nuclear magnetic resonance (NMR) spectroscopy. The NMR chemical shift perturbation and isothermal titration calorimetry (ITC) data showed that p53TAD interacted with MED25 ACID mainly through the p53TAD2 sequence motif. Taken together with the mutagenesis data, the refined structural model of MED25 ACID/p53TAD2 peptide complex showed that an amphipathic α-helix of p53TAD2 peptide bound an elongated hydrophobic groove of MED25 ACID. Furthermore, our results revealed the highly conserved mechanism of MED25 interaction with intrinsically unfolded acidic TADs from the transcriptional activators p53, ERM (Ets-related molecule), and herpes simplex virus protein 16 (VP16).
Assuntos
Complexo Mediador/química , Complexo Mediador/metabolismo , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Sítios de Ligação , Sequência Conservada , Humanos , Espectroscopia de Ressonância Magnética , Complexo Mediador/genética , Modelos Moleculares , Mutação , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/genéticaRESUMO
Anti-apoptotic proteins, like the Bcl-2 family proteins, present an important therapeutic cancer drug target. Their activity is orchestrated through neutralization upon interaction of pro-apoptotic protein counterparts that leads to immortality of cancer cells. Therefore, generating compounds targeting these proteins is of immense therapeutic importance. Herein, Induced Fit Docking (IFD) and Molecular Dynamics (MD) simulations were performed to rationally design quercetin analogues that bind in the BH3 site of the Bcl-xL protein. IFD calculations determined their binding cavity while Molecular Mechanics Poisson Boltzmann Surface Area (MM-PBSA) and Molecular Mechanics Generalised Born Surface Area (MM-GBSA) calculations provided an insight into the binding enthalpies of the analogues. The quercetin analogues were synthesized and their binding to Bcl-xL was verified with fluorescence spectroscopy. The binding affinity and the thermodynamic parameters between Bcl-xL and quercetin-glutamic acid were estimated through Isothermal Titration Calorimetry. 2D 1H-15N HSQC NMR chemical shift perturbation mapping was used to chart the binding site of the quercetin analogues in the Bcl-xL that overlapped with the predicted poses generated by both IFD and MD calculations. Furthermore, evaluation of the four conjugates against the prostate DU-145 and PC-3 cancer cell lines, revealed quercetin-glutamic acid and quercetin-alanine as the most potent conjugates bearing the higher cytostatic activity. This pinpoints that the chemical space of natural products can be tailored to exploit new hits for difficult tractable targets such as protein-protein interactions.
Assuntos
Aminoácidos/farmacologia , Antineoplásicos/farmacologia , Citostáticos/farmacologia , Desenho de Fármacos , Quercetina/farmacologia , Proteína bcl-X/antagonistas & inibidores , Aminoácidos/química , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Citostáticos/síntese química , Citostáticos/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Modelos Moleculares , Estrutura Molecular , Quercetina/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Protein tyrosine phosphatase-Basophil (PTP-Bas) is a membrane-associated protein tyrosine phosphatase with five PDZ domains and is involved in apoptosis, tumorigenesis, and insulin signaling. The interaction between PTP-Bas and tandem-PH-domain-containing protein 1/2 (TAPP1/2) plays an essential role in the regulation of insulin signaling. Despite its high sequence homology with the other PDZ domains, only the PDZ1 domain of PTP-Bas showed distinct binding specificity for TAPP1/2. Although the interaction between PTP-Bas PDZ1 and TAPP1/2 is a therapeutic target for diabetes, the structural basis for the interaction has not been elucidated. In the present study, we determined the crystal structure of the PTP-Bas PDZ1 domain at 1.6 Å resolution. In addition, we calculated the structural models of complexes of PTP-Bas PDZ1 and the C-terminal peptides of TAPP1/2 (referred to as TAPP1p/2p). Structural comparison with the PTP-Bas PDZ2/RA-GEF2 peptide complex revealed a structural basis for distinct binding specificity of PTP-Bas PDZ1 for TAPP1p/2p peptides. Our high-resolution crystal structure of PTP-Bas PDZ1 will serve as a useful template for rational structure-based design of novel anti-diabetes therapeutics.
Assuntos
Cristalografia por Raios X , Proteína Tirosina Fosfatase não Receptora Tipo 13/química , Proteína Tirosina Fosfatase não Receptora Tipo 13/metabolismo , Sequência de Aminoácidos , Humanos , Modelos Moleculares , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Domínios Proteicos , Homologia Estrutural de ProteínaRESUMO
Nanopore sensing is an emerging technology for the single-molecule-based detection of various biomolecules. In this study, we probed the anticancer therapeutic p53 transactivation domain (p53TAD)/MDM2 interaction and its inhibition with a small-molecule MDM2 antagonist, Nutlin-3, using low-noise solid-state nanopores. Although the translocation of positively charged MDM2 through a nanopore was detected at the applied negative voltage, this MDM2 translocation was almost completely blocked upon formation of the MDM2/GST-p53TAD complex owing to charge conversion. In combination with NMR data, the nanopore measurements showed that the addition of Nutlin-3 rescued MDM2 translocation, indicating that Nutlin-3 disrupted the MDM2/GST-p53TAD complex, thereby releasing MDM2. Taken together, our results reveal that solid-state nanopores can be a valuable platform for the ultrasensitive, picomole-scale screening of small-molecule drugs against protein-protein interaction (PPI) targets.
Assuntos
Antineoplásicos/química , Nanoporos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/química , Animais , Antineoplásicos/metabolismo , Compostos de Bifenilo/química , Compostos de Bifenilo/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Imidazóis/química , Imidazóis/metabolismo , Camundongos , Nitrofenóis/química , Nitrofenóis/metabolismo , Ressonância Magnética Nuclear Biomolecular , Piperazinas/química , Piperazinas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Sulfonamidas/química , Sulfonamidas/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
p73 is a member of the p53 family of transcription factors which plays an essential role in tumor suppression. p73 is associated with the sensitivity of cancer cells to chemotherapy and the prognosis of many cancers. In this study, we showed the ubiquitination-dependent degradation of p73 by the mitochondrial E3 ubiquitin ligase Hades. First, the binding between p73 and Hades was identified by co-immunoprecipitation experiments, and it was found that the Hades RING-finger domain mediates the interaction with p73. Immunofluorescence analysis showed that p73 moves to the mitochondria and colocalizes with Hades during etoposide-induced apoptosis. By performing in vivo and in vitro ubiquitination assays, we observed that the Hades RING-finger domain promotes ubiquitination of p73. Finally, it was shown that SiRNA-mediated depletion of Hades stabilizes p73. Taken together, our results showed that Hades mediates the ubiquitination-dependent degradation of mitochondrial p73 under apoptotic conditions. These findings suggest that Hades-mediated p73 ubiquitination is a novel regulatory mechanism for the exonuclear function of p73.
Assuntos
Proteínas de Ligação a DNA/genética , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Mitocôndrias/metabolismo , Proteínas Nucleares/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Sítios de Ligação , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Etoposídeo/farmacologia , Humanos , Pulmão/metabolismo , Pulmão/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteólise , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteína Tumoral p73 , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Molecular interactions between the tumor suppressor p53 and the anti-apoptotic Bcl-2 family proteins play an important role in the transcription-independent apoptosis of p53. The p53 transactivation domain (p53TAD) contains two conserved ΦXXΦΦ motifs (Φ indicates a bulky hydrophobic residue and X is any other residue) referred to as p53TAD1 (residues 15-29) and p53TAD2 (residues 39-57). We previously showed that p53TAD1 can act as a binding motif for anti-apoptotic Bcl-2 family proteins. In this study, we have identified p53TAD2 as a binding motif for anti-apoptotic Bcl-2 family proteins by using NMR spectroscopy, and we calculated the structures of Bcl-X(L)/Bcl-2 in complex with the p53TAD2 peptide. NMR chemical shift perturbation data showed that p53TAD2 peptide binds to diverse members of the anti-apoptotic Bcl-2 family independently of p53TAD1, and the binding between p53TAD2 and p53TAD1 to Bcl-X(L) is competitive. Refined structural models of the Bcl-X(L)·p53TAD2 and Bcl-2·p53TAD2 complexes showed that the binding sites occupied by p53TAD2 in Bcl-X(L) and Bcl-2 overlap well with those occupied by pro-apoptotic BH3 peptides. Taken together with the mutagenesis, isothermal titration calorimetry, and paramagnetic relaxation enhancement data, our structural comparisons provided the structural basis of p53TAD2-mediated interaction with the anti-apoptotic proteins, revealing that Bcl-X(L)/Bcl-2, MDM2, and cAMP-response element-binding protein-binding protein/p300 share highly similar modes of binding to the dual p53TAD motifs, p53TAD1 and p53TAD2. In conclusion, our results suggest that the dual-site interaction of p53TAD is a highly conserved mechanism underlying target protein binding in the transcription-dependent and transcription-independent apoptotic pathways of p53.
Assuntos
Apoptose , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteína Supressora de Tumor p53/química , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Sítios de Ligação/genética , Ligação Competitiva , Calorimetria , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Ativação Transcricional , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína bcl-X/química , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Inhibition of the interaction between the p53 tumor suppressor and its negative regulator MDM2 is of great importance to cancer therapy. The anti-apoptotic Bcl-2 family proteins are also attractive anti-cancer molecular targets, as they are key regulators of apoptotic cell death. Previously, we reported the interactions between the p53 transactivation domain (p53TAD) and diverse members of the anti-apoptotic Bcl-2 family proteins. In this study, we investigated the binding of MDM2-inhibiting p53TAD peptide analogues, p53-MDM2/MDMX inhibitor (PMI) and pDI, with anti-apoptotic Bcl-2 family proteins, Bcl-XL and Bcl-2, by using NMR spectroscopy. The NMR chemical shift perturbation data demonstrated the direct binding of the p53 peptide analogues to Bcl-XL and Bcl-2 and showed that the PMI and pDI peptides bind to a conserved hydrophobic groove of the anti-apoptotic Bcl-2 family proteins. Furthermore, the structural model of the Bcl-XL/PMI peptide complex showed that the binding mode of the PMI peptide is highly similar to that of pro-apoptotic Bcl-2 homology 3 (BH3) peptides. Finally, our structural comparison provided a molecular basis for how the same PMI peptide can bind to two distinct anti-cancer target proteins Bcl-XL and MDM2, which may have potential applications for multi-targeting cancer therapy.
Assuntos
Apoptose , Espectroscopia de Ressonância Magnética , Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína bcl-X/química , Proteína bcl-X/metabolismoRESUMO
The interaction between tumor suppressor p53 and the anti-apoptotic Bcl-2 family proteins serves a critical role in the transcription-independent apoptosis mechanism of p53. Our previous studies showed that an MDM2-inhibiting motif (residues 15-29) in the p53 transactivation domain (p53TAD) mediates the interaction with anti-apoptotic Bcl-2 family proteins. In this study, we provided structural models of the complexes between the MDM2-inhibiting p53TAD peptide and Mcl-1, Bcl-w, and Kaposi sarcoma-associated herpes virus (KSHV) Bcl-2 using NMR chemical shift perturbation data. The binding mode of the MDM2-inhibiting p53TAD peptide is highly conserved among the anti-apoptotic Bcl-2 family proteins despite their distinct specificities for pro-apoptotic Bcl-2 family proteins. We also identified the binding of a phage-display-derived MDM2-inhibiting peptide 12-1 to anti-apoptotic Bcl-XL protein by using NMR spectroscopy. The structural model of the Bcl-XL/12-1 peptide complex revealed that the conserved residues Phe4, Trp8, and Leu11 in the MDM2-inhibiting peptide fit into a hydrophobic cleft of Bcl-XL in a manner similar to that of pro-apoptotic Bcl-2 homology 3 (BH3) peptides. Our results shed light on the mechanism underlying dual-targeting of the FxxxWxxL-based α-helical motif to MDM2 and anti-apoptotic Bcl-2 family proteins for anticancer therapy.
Assuntos
Peptídeos/química , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-mdm2/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Sítios de Ligação , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Oncogênicas/química , Proteínas Oncogênicas/metabolismo , Peptídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteína bcl-X/química , Proteína bcl-X/metabolismoRESUMO
Dual-specificity phosphatases (DUSPs) play an important role in regulating cellular signalling pathways governing cell growth, differentiation and apoptosis. Human DUSP26 inhibits the apoptosis of cancer cells by dephosphorylating substrates such as p38 and p53. High-resolution crystal structures of the DUSP26 catalytic domain (DUSP26-C) and its C152S mutant [DUSP26-C (C152S)] have been determined at 1.67 and 2.20 Å resolution, respectively. The structure of DUSP26-C showed a novel type of domain-swapped dimer formed by extensive crossover of the C-terminal α7 helix. Taken together with the results of a phosphatase-activity assay, structural comparison with other DUSPs revealed that DUSP26-C adopts a catalytically inactive conformation of the protein tyrosine phosphate-binding loop which significantly deviates from that of canonical DUSP structures. In particular, a noticeable difference exists between DUSP26-C and the active forms of other DUSPs at the hinge region of a swapped C-terminal domain. Additionally, two significant gaps were identified between the catalytic core and its surrounding loops in DUSP26-C, which can be exploited as additional binding sites for allosteric enzyme regulation. The high-resolution structure of DUSP26-C may thus provide structural insights into the rational design of DUSP26-targeted anticancer drugs.
Assuntos
Fosfatases de Especificidade Dupla/química , Fosfatases da Proteína Quinase Ativada por Mitógeno/química , Proteínas Mutantes/química , Calorimetria , Domínio Catalítico , Fosfatases de Especificidade Dupla/genética , Fosfatases de Especificidade Dupla/metabolismo , Humanos , Fosfatases da Proteína Quinase Ativada por Mitógeno/genética , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Mutagênese , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Difração de Raios XRESUMO
The COVID-19 pandemic is an ongoing global public health threat. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and binding of the SARS-CoV-2 spike to its receptor, angiotensin-converting enzyme 2 (ACE2), on host cells is critical for viral infection. Here, we developed a luminescent biosensor that readily detects interactions of the spike receptor-binding domain (RBD) and ACE2 in cell culture medium ('SpACE-CCM'), which was based on bimolecular complementation of the split nanoluciferase-fused spike RBD and ectodomain of ACE2 and further engineered to be efficiently secreted from cells by adding a heterologous secretory signal peptide (SSP). Screening of various SSPs identified 'interferon-α+alanine-aspartate' as the SSP that induced the highest activity. The SpACE-CCM biosensor was validated by observing a marked reduction of the activity caused by interaction-defective mutations or in the presence of neutralizing antibodies, recombinant decoy proteins, or peptides. Importantly, the SpACE-CCM biosensor responded well in assay-validating conditions compared with conventional cell lysate-based NanoLuc Binary Technology, indicating its advantage. We further demonstrated the biosensor's versatility by quantitatively detecting neutralizing activity in blood samples from COVID-19 patients and vaccinated individuals, discovering a small molecule interfering with the spike RBD-ACE2 interaction through high-throughput screening, and assessing the cross-reactivity of neutralizing antibodies against SARS-CoV-2 variants. Because the SpACE-CCM is a facile and rapid one-step reaction biosensor that aptly recapitulates the native spike-ACE2 interaction, it would be advantageous in many experimental and clinical applications associated with this interaction.
Assuntos
Técnicas Biossensoriais , COVID-19 , Humanos , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2 , Pandemias , Ligação Proteica , Anticorpos Neutralizantes/metabolismo , Técnicas de Cultura de Células , Glicoproteína da Espícula de CoronavírusRESUMO
In drug discovery, efficient screening of protein-drug interactions (PDIs) is hampered by the limitations of current biophysical approaches. Here, we develop a biological nanopore sensor for single-molecule detection of proteins and PDIs using the pore-forming toxin YaxAB. Using this YaxAB nanopore, we demonstrate label-free, single-molecule detection of interactions between the anticancer Bcl-xL protein and small-molecule drugs as well as the Bak-BH3 peptide. The long funnel-shaped structure and nanofluidic characteristics of the YaxAB nanopore enable the electro-osmotic trapping of diverse folded proteins and high-resolution monitoring of PDIs. Distinctive nanopore event distributions observed in the two-dimensional (ΔI/Io-versus-IN) plot illustrate the ability of the YaxAB nanopore to discriminate individual small-molecule drugs bound to Bcl-xL from non-binders. Taken together, our results present the YaxAB nanopore as a robust platform for label-free, ultrasensitive, single-molecule detection of PDIs, opening up a possibility for low-cost, highly efficient drug discovery against diverse drug targets.