RESUMEN
Upon binding to DNA breaks, poly(ADP-ribose) polymerase 1 (PARP1) ADP-ribosylates itself and other factors to initiate DNA repair. Serine is the major residue for ADP-ribosylation upon DNA damage, which strictly depends on HPF1. Here, we report the crystal structures of human HPF1/PARP1-CAT ΔHD complex at 1.98 Å resolution, and mouse and human HPF1 at 1.71 Å and 1.57 Å resolution, respectively. Our structures and mutagenesis data confirm that the structural insights obtained in a recent HPF1/PARP2 study by Suskiewicz et al. apply to PARP1. Moreover, we quantitatively characterize the key residues necessary for HPF1/PARP1 binding. Our data show that through salt-bridging to Glu284/Asp286, Arg239 positions Glu284 to catalyze serine ADP-ribosylation, maintains the local conformation of HPF1 to limit PARP1 automodification, and facilitates HPF1/PARP1 binding by neutralizing the negative charge of Glu284. These findings, along with the high-resolution structural data, may facilitate drug discovery targeting PARP1.
Asunto(s)
Proteínas Portadoras/química , ADN/química , Histonas/química , Proteínas Nucleares/química , Poli(ADP-Ribosa) Polimerasa-1/química , Serina/metabolismo , ADP-Ribosilación , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Clonación Molecular , Cristalografía por Rayos X , ADN/genética , ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glutamina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Ratones , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Electricidad EstáticaRESUMEN
Tumor necrosis factor-α (TNF-α) is a pluripotent mediator of pro-inflammatory and antimicrobial defense mechanisms and a regulator of lymphoid organ development. Although two types of TNF-α have been identified in several teleost species, their functions in pathogen infection remain largely unexplored, especially in pathogen clearance. Herein, we cloned and characterized two types of TNF-α, termed shTNF-α1 and shTNF-α2, and their receptors, shTNFR1 and shTNFR2, from snakehead (Channa argus). These genes were constitutively expressed in all tested tissues, and were induced by Aeromonas schubertii and Nocardia seriolae in head kidney and spleen in vivo, and by lipoteichoic acid (LTA), lipopolysaccharides (LPS), and Polyinosinic-polycytidylic acid [Poly (I:C)] in head kidney leukocytes (HKLs) in vitro. Moreover, recombinant shTNF-α1 and shTNF-α2 upregulated the expression of endogenous shTNF-α1, shTNF-α2, shTNFR1, and shTNFR2, and enhanced intracellular bactericidal activity, with shTNF-α1 having a greater effect than shTNF-α2. These findings suggest important roles of fish TNFα1, TNFα2, and their receptors in bacterial infection and pathogen clearance, and provide a new insight into their function in antibacterial innate immunity.
Asunto(s)
Enfermedades de los Peces/inmunología , Peces/genética , Peces/inmunología , Inmunidad Innata/genética , Receptores del Factor de Necrosis Tumoral/genética , Factor de Necrosis Tumoral alfa/genética , Aeromonas/fisiología , Animales , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Riñón Cefálico/inmunología , Leucocitos/inmunología , Lipopolisacáridos/farmacología , Nocardia/fisiología , Nocardiosis/inmunología , Nocardiosis/veterinaria , Poli I-C/farmacología , Receptores del Factor de Necrosis Tumoral/inmunología , Bazo/inmunología , Ácidos Teicoicos/farmacología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
As a central pro-inflammatory cytokine, interleukin-1ß (IL-1ß) plays critical roles in the inflammatory response, pathogen infection, and immunological challenges in mammals. Although fish IL-1ß has been confirmed to participate in inflammatory response to pathogen infection, few studies have been performed to characterize the antibacterial and bactericidal functions of fish IL-1ß. In this study, snakehead (Channa argus) IL-1ß (shIL-1ß) and its receptors, shIL-1R1 and shIL-1R2, were cloned and functionally characterized. ShIL-1ß contained the IL-1 family signature domain, and a potential cutting site at Asp96 that presented in all vertebrate IL-1ß sequences. ShIL-1R1 had three extracellular IG-like domains and one intracellular signal TIR domain, while shIL-1R2 had three extracellular IG-like domain but lacked the intracellular signal TIR domain. ShIL-1ß, shIL-1R1, and shIL-1R2 were constitutively expressed in all tested tissues, and their expressions could be induced by Aeromonas schubertii and Nocardia seriolae in the head kidney and spleen in vivo, and by LTA, LPS, and Poly (I:C) in head kidney leukocytes (HKLs) in vitro. Moreover, recombinant shIL-1ß upregulated the expression of endogenous shIL-1ß, shIL-R1, and shIL-R2 in snakehead HKLs, and enhanced intracellular bactericidal activity. Taken together, this study found that, like IL-1ß and its receptors in mammals, shIL-1ß and its receptors play crucial roles in antibacterial innate immunity. This provides new insight into the evolution of IL-1ß function in vertebrates.
Asunto(s)
Bacterias/inmunología , Infecciones Bacterianas/veterinaria , Carpas/inmunología , Enfermedades de los Peces/inmunología , Inmunidad Innata , Interleucina-1beta/genética , Receptores de Interleucina-1/genética , Animales , Antibacterianos , Infecciones Bacterianas/inmunología , Carpas/genética , Carpas/microbiología , Clonación Molecular , Enfermedades de los Peces/microbiología , Riñón Cefálico/inmunología , Interleucina-1beta/inmunología , Receptores de Interleucina-1/inmunologíaRESUMEN
Drug-resistance is a major challenge in targeted therapy of EGFR mutated non-small cell lung cancers (NSCLCs). The third-generation irreversible inhibitors such as AZD9291, CO-1686 and WZ4002 can overcome EGFR T790M drug-resistance mutant through covalent binding through Cys 797, but ultimately lose their efficacy upon emergence of the new mutation C797S. To develop new reversible inhibitors not relying on covalent binding through Cys 797 is therefore urgently demanded. Gö6976 is a staurosporine-like reversible inhibitor targeting T790M while sparing the wild-type EGFR. In the present work, we reported the complex crystal structures of EGFR T790M/C797S + Gö6976 and T790M + Gö6976, along with enzyme kinetic data of EGFR wild-type, T790M and T790M/C797S. These data showed that the C797S mutation does not significantly alter the structure and function of the EGFR kinase, but increases the local hydrophilicity around residue 797. The complex crystal structures also elucidated the detailed binding mode of Gö6976 to EGFR and explained why this compound prefers binding to T790M mutant. These structural pharmacological data would facilitate future drug development studies.
Asunto(s)
Carbazoles/farmacología , Receptores ErbB/química , Receptores ErbB/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Carbazoles/química , Relación Dosis-Respuesta a Droga , Receptores ErbB/genética , Humanos , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/química , Relación Estructura-ActividadRESUMEN
Tissue repair and regenerative medicine address the important medical needs to replace damaged tissue with functional tissue. Most regenerative medicine strategies have focused on delivering biomaterials and cells, yet there is the untapped potential for drug-induced regeneration with good specificity and safety profiles. The Hippo pathway is a key regulator of organ size and regeneration by inhibiting cell proliferation and promoting apoptosis. Kinases MST1 and MST2 (MST1/2), the mammalian Hippo orthologs, are central components of this pathway and are, therefore, strong target candidates for pharmacologically induced tissue regeneration. We report the discovery of a reversible and selective MST1/2 inhibitor, 4-((5,10-dimethyl-6-oxo-6,10-dihydro-5H-pyrimido[5,4-b]thieno[3,2-e][1,4]diazepin-2-yl)amino)benzenesulfonamide (XMU-MP-1), using an enzyme-linked immunosorbent assay-based high-throughput biochemical assay. The cocrystal structure and the structure-activity relationship confirmed that XMU-MP-1 is on-target to MST1/2. XMU-MP-1 blocked MST1/2 kinase activities, thereby activating the downstream effector Yes-associated protein and promoting cell growth. XMU-MP-1 displayed excellent in vivo pharmacokinetics and was able to augment mouse intestinal repair, as well as liver repair and regeneration, in both acute and chronic liver injury mouse models at a dose of 1 to 3 mg/kg via intraperitoneal injection. XMU-MP-1 treatment exhibited substantially greater repopulation rate of human hepatocytes in the Fah-deficient mouse model than in the vehicle-treated control, indicating that XMU-MP-1 treatment might facilitate human liver regeneration. Thus, the pharmacological modulation of MST1/2 kinase activities provides a novel approach to potentiate tissue repair and regeneration, with XMU-MP-1 as the first lead for the development of targeted regenerative therapeutics.
Asunto(s)
Factor de Crecimiento de Hepatocito/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Regeneración/efectos de los fármacos , Sulfonamidas/farmacología , Acetaminofén/toxicidad , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Colitis/inducido químicamente , Colitis/prevención & control , Cristalización , Factor de Crecimiento de Hepatocito/química , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/trasplante , Ensayos Analíticos de Alto Rendimiento , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lesión Pulmonar/tratamiento farmacológico , Ratones , Modelos Moleculares , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/química , Medicina Regenerativa , Serina-Treonina Quinasa 3 , Transducción de Señal/efectos de los fármacos , Sulfonamidas/química , Investigación Biomédica TraslacionalRESUMEN
BCR gene fused ABL kinase is the critical driving force for the Philadelphia Chromosome positive (Ph+) Chronic Myeloid Leukemia (CML) and has been extensively explored as a drug target. With a structure-based drug design approach we have discovered a novel inhibitor CHMFL-074, that potently inhibits both the native and a variety of clinically emerged mutants of BCR-ABL kinase. The X-ray crystal structure of CHMFL-074 in complex with ABL1 kinase (PDB ID: 5HU9) revealed a typical type II binding mode (DFG-out) but relatively rare hinge binding. Kinome wide selectivity profiling demonstrated that CHMFL-074 bore a high selectivity (S score(1) = 0.03) and potently inhibited ABL1 kinase (IC50: 24 nM) and PDGFR α/ß (IC50: 71 nM and 88 nM). CHMFL-074 displayed strong anti-proliferative efficacy against BCR-ABL-driven CML cell lines such as K562 (GI50: 56 nM), MEG-01 (GI50: 18 nM) and KU812 (GI50: 57 nM). CHMFL-074 arrested cell cycle into the G0/G1 phase and induced apoptosis in the Ph+ CML cell lines. In addition, it potently inhibited the CML patient primary cell's proliferation but did not affect the normal bone marrow cells. In the CML cell K562 inoculated xenograft mouse model, oral administration of 100 mg/kg/d of CHMFL-074 achieved a tumor growth inhibition (TGI) of 65% without exhibiting apparent toxicity. As a potential drug candidate for fighting CML, CHMFL-074 is under extensive preclinical safety evaluation now.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Animales , Antineoplásicos/química , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Biochar is a carbon-rich material derived from incomplete combustion of biomass.Applying biochar as an amendment to treat contaminated soils is receiving increasing attention, and is a promising way to improve soil quality. Heavy metals are persistent and are not environmentally biodegradable. However, they can be stabilized in soil by adding biochar. Moreover, biochar is considered to be a predominant sorptive agent for organic pollutants, having a removal efficiency of about 1 order of magnitude higher than does soil/sediment organic matter or their precursor substances alone.When trying to stabilize organic and inorganic pollutants in soil, several features of biochar' s sorption capacity should be considered, viz., the nature of the pollutants to be remediated, how the biochar is prepared, and the complexity of the soil systemin which biochar may be used. In addition, a significant portion of the biochar or some of its components that are used to remediate soils do change over time through abiotic oxidation and microbial decomposition. This change process is commonly referred to as "aging:" Biochar "aging" in nature is inevitable, and aged biochar exhibits an effect that is totally different than non-aged biochar on stabilizing heavy metals and organic contaminants in soils.Studies that have been performed to date on the use of biochar to remediate contaminated soil are insufficient to allow its use for wide-scale field application.Therefore, considerable new data are necessary to expand both our understanding of how biochar performs in the field, and where it can be best used in the future for soil remediation. For example, how biochar and soil biota (microbial and faunal communities)interact in soils is still poorly understood. Moreover, studies are needed on how to best remove new species of heavy metals, and on how biochar aging affects sorption capacity are also needed.