RESUMEN
BACKGROUND: Many esophageal striated muscles of mammals are dually innervated by the vagal and enteric nerves. Recently, substance P (SP)-sensory nerve terminals with calcitonin gene-related peptide (CGRP) were found on a few striated muscle fibers in the rat esophagus, implying that these muscle fibers are triply innervated. In this study, we examined the localization and origin of CGRP-nerve endings in striated muscles to consider their possible roles in the esophagus regarding triple innervation. METHODS: Wholemounts of the rat esophagus were immunolabeled to detect CGRP-nerve endings in striated muscles. Also, retrograde tracing was performed by injecting Fast Blue (FB) into the esophagus, and cryostat sections of the medulla oblongata, nodose ganglion (NG), and the tenth thoracic (T10) dorsal root ganglion (DRG) were immunostained to identify the origin of the CGRP-nerve endings. RESULTS: CGRP-fine, varicose nerve endings were localized in motor endplates on a few esophageal striated muscle fibers (4 %), most of which received nitric oxide (NO) synthase nerve terminals, and most of the CGRP nerve endings were SP- and transient receptor potential vanilloid member 1 (TRPV1)-positive. Retrograde tracing showed many FB-labeled CGRP-neurons positive for SP and TRPV1 in the NG and T10 DGR. CONCLUSIONS: This study suggests that the CGRP-varicose nerve endings containing SP and TRPV1 in motor endplates are sensory, and a few esophageal striated muscle fibers are triply innervated. The nerve endings may detect acetylcholine-derived acetic acid from the vagal motor nerve endings and NO from esophageal intrinsic nerve terminals in the motor endplates to regulate esophageal motility.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina , Esófago , Ganglio Nudoso , Células Receptoras Sensoriales , Animales , Péptido Relacionado con Gen de Calcitonina/metabolismo , Péptido Relacionado con Gen de Calcitonina/análisis , Esófago/inervación , Esófago/metabolismo , Masculino , Células Receptoras Sensoriales/metabolismo , Ganglio Nudoso/metabolismo , Placa Motora/metabolismo , Ratas , Ganglios Espinales/metabolismo , Bulbo Raquídeo/metabolismo , Sustancia P/metabolismo , Músculo Estriado/inervación , Músculo Estriado/metabolismo , Nervio Vago/metabolismo , Ratas Wistar , Ratas Sprague-Dawley , Fibras Musculares Esqueléticas/metabolismo , Canales Catiónicos TRPV/metabolismo , AmidinasRESUMEN
A chemical proteomics approach using Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor-immobilized sepharose (TIM-063-Kinobeads) identified main targets such as CaMKKα/1 and ß/2, and potential off-target kinases, including AP2-associated protein kinase 1 (AAK1), as TIM-063 interactants. Because TIM-063 interacted with the AAK1 catalytic domain and inhibited its enzymatic activity moderately (IC50 = 8.51 µM), we attempted to identify potential AAK1 inhibitors from TIM-063-derivatives and found a novel AAK1 inhibitor, TIM-098a (11-amino-2-hydroxy-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) which is more potent (IC50 = 0.24 µM) than TIM-063 without any inhibitory activity against CaMKK isoforms and a relative AAK1-selectivity among the Numb-associated kinases family. TIM-098a could inhibit AAK1 activity in transfected cultured cells (IC50 = 0.87 µM), indicating cell-membrane permeability of the compound. Overexpression of AAK1 in HeLa cells significantly reduced the number of early endosomes, which was blocked by treatment with 10 µM TIM-098a. These results indicate TIM-063-Kinobeads-based chemical proteomics is efficient for identifying off-target kinases and re-evaluating the kinase inhibitor (TIM-063), leading to the successful development of a novel inhibitory compound (TIM-098a) for AAK1, which could be a molecular probe for AAK1. TIM-098a may be a promising lead compound for a more potent, selective and therapeutically useful AAK1 inhibitor.
Asunto(s)
Inhibidores de Proteínas Quinasas , Proteínas Serina-Treonina Quinasas , Humanos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Células HeLa , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , FosforilaciónRESUMEN
Histone variants play a central role in shaping the chromatin landscape in plants, yet, how their distinct combinations affect nucleosome properties and dynamics is still largely elusive. To address this, we developed a novel chromatin assembly platform for Arabidopsis thaliana, using wheat germ cell-free protein expression. Four canonical histones and five reported histone variants were used to assemble twelve A. thaliana nucleosome combinations. Seven combinations were successfully reconstituted and confirmed by supercoiling and micrococcal nuclease (MNase) assays. The effect of the remodeling function of the CHR11-DDR4 complex on these seven combinations was evaluated based on the nucleosome repeat length and nucleosome spacing index obtained from the MNase ladders. Overall, the current study provides a novel method to elucidate the formation and function of a diverse range of nucleosomes in plants.
Asunto(s)
Arabidopsis , Nucleosomas , Nucleosomas/genética , Ensamble y Desensamble de Cromatina , Histonas/genética , Cromatina/genética , Arabidopsis/genéticaRESUMEN
HIV-1 Vif is known to counteract the antiviral activity of human apolipoprotein B mRNA-editing catalytic polypeptide-like (A3), a cytidine deaminase, in various ways. However, the precise mechanism behind this interaction has remained elusive. Within infected cells, Vif forms a complex called VßBCC, comprising CBFß and the components of E3 ubiquitin ligase, Elongin B, Elongin C, and Cullin5. Together with the ubiquitin-conjugating enzyme, VßBCC induces ubiquitination-mediated proteasomal degradation of A3. However, Vif exhibits additional counteractive effects. In this study, we elucidate that VßBCC inhibits deamination by A3G, A3F, and A3B independently of proteasomal degradation. Surprisingly, we discovered that this inhibition for A3G is directly attributed to the interaction between VßBCC and the C-terminal domain of A3G. Previously, it was believed that Vif did not interact with the C-terminal domain. Our findings suggest that inhibiting the interaction between VßBCC and the C-terminal domain, as well as the N-terminal domain known to be targeted for ubiquitination, of A3G may be needed to prevent counteraction by Vif.
Asunto(s)
VIH-1 , Productos del Gen vif del Virus de la Inmunodeficiencia Humana , Humanos , Citosina Desaminasa/metabolismo , VIH-1/metabolismo , Unión Proteica , ProteolisisRESUMEN
Given that the current approved anti-hepatitis B virus (HBV) drugs suppress virus replication and improve hepatitis but cannot eliminate HBV from infected patients, new anti-HBV agents with different mode of action are urgently needed. In this study, we identified a semi-synthetic oxysterol, Oxy185, that can prevent HBV infection in a HepG2-based cell line and primary human hepatocytes. Mechanistically, Oxy185 inhibited the internalization of HBV into cells without affecting virus attachment or replication. We also found that Oxy185 interacted with an HBV entry receptor, sodium taurocholate cotransporting polypeptide (NTCP), and inhibited the oligomerization of NTCP to reduce the efficiency of HBV internalization. Consistent with this mechanism, Oxy185 also inhibited the hepatitis D virus infection, which relies on NTCP-dependent internalization, but not hepatitis A virus infection, and displayed pan-genotypic anti-HBV activity. Following oral administration in mice, Oxy185 showed sustained accumulation in the livers of the mice, along with a favorable liver-to-plasma ratio. Thus, Oxy185 is expected to serve as a useful tool compound in proof-of-principle studies for HBV entry inhibitors with this novel mode of action.
Asunto(s)
Hepatitis B , Simportadores , Humanos , Ratones , Animales , Virus de la Hepatitis B/fisiología , Internalización del Virus , Hepatitis B/metabolismo , Hepatocitos/metabolismo , Células Hep G2 , Virus de la Hepatitis Delta/metabolismo , Simportadores/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismoRESUMEN
Calmodulin (CaM) is known to function as a central signal transducer in calcium-mediated intracellular pathways. In this study, a fusion molecule of a recently developed proximity biotinylation enzyme (AirID) with rat CaM (AirID-CaM) was expressed and purified to near homogeneity using an E. coli expression system to examine the physical interactions between CaM and its target proteins by converting the interaction to biotinylation of CaM targets under nondenatured conditions. AirID-CaM catalyzed a Ca2+-dependent biotinylation of a target protein kinase (Ca2+/CaM-dependent protein kinase kinase α/1, CaMKKα/1) in vitro, which was suppressed by the addition of excess amounts of CaM, and AirID alone did not catalyze the biotinylation of CaMKKα/1, indicating that the biotinylation of CaMKKα/1 by AirID-CaM likely occurs in an interaction-dependent manner. Furthermore, we also observed the Ca2+-dependent biotinylation of GST-CaMKIα and GST-CaMKIV by AirID-CaM, suggesting that AirID-CaM can be useful for the rapid detection of CaM/target interactions with relatively high sensitivity.
Asunto(s)
Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina , Calmodulina , Ratas , Animales , Calmodulina/metabolismo , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Biotinilación , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Fosforilación , Calcio/metabolismoRESUMEN
The present immunohistochemical study was performed to examine the number, distribution, and chemical coding of intrinsic substance P (SP) neurons and nerve fibers within the esophagus and discuss their functional roles. Many SP neurons and nerve fibers were found in the myenteric plexus, and the SP neurons gradually decreased from the oral side toward the aboral side of the esophagus. Double-immunolabeling showed that most SP neurons were cholinergic (positive for choline acetyltransferase), and few were nitrergic (positive for nitric oxide synthase). Some cholinergic SP nerve terminals surrounded cell bodies of several myenteric neurons. In the muscularis mucosa and lower esophageal sphincter, and around blood vessels, numerous SP nerve endings were present, and many of them were cholinergic. Also, SP nerve endings were found on only a few motor endplates of the striated muscles, and most of them were calcitonin gene-related peptide (CGRP)-positive. Retrograde tracing using Fast Blue (FB) showed that numerous sensory neurons in the dorsal root ganglia (DRGs) and nodose ganglion (NG) projected to the esophagus, and most FB-labeled SP neurons were CGRP-positive. These results suggest that the intrinsic SP neurons in the rat esophagus may play roles as, at least, motor neurons, interneurons, and vasomotor neurons, which are involved in local regulation of smooth muscle motility, neuronal transmission, and blood circulation, respectively. Moreover, SP nerve endings on only a minority of motor endplates may be extrinsic, derived from DRGs or NG, and possibly detect chemical circumstances within motor endplates to modulate esophageal motility.
Asunto(s)
Plexo Mientérico , Sustancia P , Ratas , Animales , Péptido Relacionado con Gen de Calcitonina , Neuronas Motoras , EsófagoRESUMEN
Protein-protein interaction (PPI) analysis is a key process to understand protein functions. Recently, we constructed a human protein array (20 K human protein beads array) consisting of 19,712 recombinant human proteins produced by a wheat cell-free protein production system. Here, we developed a cell-free protein array technology for proximity biotinylation-based PPI identification (CF-PPiD). The proximity biotinylation enzyme AirID-fused TP53 and -IκBα proteins each biotinylated specific interacting proteins on a 1536-well magnetic plate. In addition, AirID-fused cereblon was shown to have drug-inducible PPIs using CF-PPiD. Using the human protein beads array with AirID-IκBα, 132 proteins were biotinylated, and then selected clones showed these biological interactions in cells. Although ZBTB9 was not immunoprecipitated, it was highly biotinylated by AirID-IκBα, suggesting that this system detected weak interactions. These results indicated that CF-PPiD is useful for the biochemical identification of directly interacting proteins.
Asunto(s)
Análisis por Matrices de Proteínas , Mapeo de Interacción de Proteínas , Biotinilación , Humanos , Inhibidor NF-kappaB alfa , Mapeo de Interacción de Proteínas/métodos , Proteínas RecombinantesRESUMEN
DNA cytosine deaminase APOBEC3B (A3B) is an endogenous source of mutations in many human cancers, including multiple myeloma. A3B proteins form catalytically inactive high molecular mass (HMM) complexes in nuclei, however, the regulatory mechanisms of A3B deaminase activity in HMM complexes are still unclear. Here, we performed mass spectrometry analysis of A3B-interacting proteins from nuclear extracts of myeloma cell lines and identified 30 putative interacting proteins. These proteins are involved in RNA metabolism, including RNA binding, mRNA splicing, translation, and regulation of gene expression. Except for SAFB, these proteins interact with A3B in an RNA-dependent manner. Most of these interacting proteins are detected in A3B HMM complexes by density gradient sedimentation assays. We focused on two interacting proteins, ILF2 and SAFB. We found that overexpressed ILF2 enhanced the deaminase activity of A3B by 30%, while SAFB did not. Additionally, siRNA-mediated knockdown of ILF2 suppressed A3B deaminase activity by 30% in HEK293T cell lysates. Based on these findings, we conclude that ILF2 can interact with A3B and enhance its deaminase activity in HMM complexes.
Asunto(s)
Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Regulación Enzimológica de la Expresión Génica/genética , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/metabolismo , Mieloma Múltiple/genética , Mutación/genética , Proteína del Factor Nuclear 45/genética , Proteína del Factor Nuclear 45/fisiología , Línea Celular Tumoral , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Proteína del Factor Nuclear 45/metabolismo , Mapas de Interacción de Proteínas/genéticaRESUMEN
To elucidate S100 protein-mediated signaling pathways, we attempted to identify novel binding partners for S100A2 by screening protein arrays carrying 19,676 recombinant glutathione S-transferase (GST)-fused human proteins with biotinylated S100A2. Among newly discovered putative S100A2 interactants, including TMLHE, TRH, RPL36, MRPS34, CDR2L, OIP5, and MED29, we identified and characterized the tubulin polymerization-promoting protein (TPPP) as a novel S100A2-binding protein. We confirmed the interaction of TPPP with Ca2+/S100A2 by multiple independent methods, including the protein array method, S100A2 overlay, and pulldown assay in vitro and in transfected COS-7 cells. Based on the results from the S100A2 overlay assay using various GST-TPPP mutants, the S100A2-binding region was identified in the C-terminal (residues 111-160) of the central core domain of a monomeric form of TPPP that is involved in TPPP dimerization. Chemical cross-linking experiments indicated that S100A2 suppresses dimer formation of His-tagged TPPP in a dose-dependent and a Ca2+-dependent manner. In addition to S100A2, TPPP dimerization is disrupted by other multiple S100 proteins, including S100A6 and S100B, in a Ca2+-dependent manner but not by S100A4. This is consistent with the fact that S100A6 and S100B, but not S100A4, are capable of interacting with GST-TPPP in the presence of Ca2+. Considering these results together, TPPP was identified as a novel target for S100A2, and it is a potential binding target for other multiple S100 proteins, including S100A6 and S100B. Direct binding of the S100 proteins with TPPP may cause disassembly of TPPP dimer formation in response to the increasing concentration of intracellular Ca2+, thus resulting in the regulation of the physiological function of TPPP, such as microtubule organization.
Asunto(s)
Calcio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Polimerizacion , Proteínas S100/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Células COS , Chlorocebus aethiops , Humanos , Proteínas del Tejido Nervioso/química , Proteínas S100/química , Tubulina (Proteína)/químicaRESUMEN
During screening of protein-protein interactions, using human protein arrays carrying 19,676 recombinant glutathione s-transferase (GST)-fused human proteins, we identified the high-mobility protein group 20A (HMG20A) as a novel S100A6 binding partner. We confirmed the Ca2+-dependent interaction of HMG20A with S100A6 by the protein array method, biotinylated S100A6 overlay, and GST-pulldown assay in vitro and in transfected COS-7 cells. Co-immunoprecipitation of S100A6 with HMG20A from HeLa cells in a Ca2+-dependent manner revealed the physiological relevance of the S100A6/HMG20A interaction. In addition, HMG20A has the ability to interact with S100A1, S100A2, and S100B in a Ca2+-dependent manner, but not with S100A4, A11, A12, and calmodulin. S100A6 binding experiments using various HMG20A mutants revealed that Ca2+/S100A6 interacts with the C-terminal region (residues 311-342) of HMG20A with stoichiometric binding (HMG20A:S100A6 dimer = 1:1). This was confirmed by the fact that a GST-HMG20A mutant lacking the S100A6 binding region (residues 311-347, HMG20A-ΔC) failed to interact with endogenous S100A6 in transfected COS-7 cells, unlike wild-type HMG20A. Taken together, these results identify, for the first time, HMG20A as a target of Ca2+/S100 proteins, and may suggest a novel linkage between Ca2+/S100 protein signaling and HMG20A function, including in the regulation of neural differentiation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteína A6 de Unión a Calcio de la Familia S100/metabolismo , Animales , Sitios de Unión , Células COS , Proteínas de Ciclo Celular/genética , Chlorocebus aethiops , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Células HeLa , Proteínas del Grupo de Alta Movilidad/genética , Humanos , Análisis por Matrices de Proteínas , Dominios y Motivos de Interacción de Proteínas , Proteína A6 de Unión a Calcio de la Familia S100/genéticaRESUMEN
Collagenase from the Grimontia hollisae strain 1706B (Ghcol) is a zinc metalloproteinase with the zinc-binding motif H492EXXH496. It exhibits higher collagen-degrading activity than the collagenase from Clostridium histolyticum, which is widely used in industry. We previously examined the pH and temperature dependencies of Ghcol activity; Glu493 was thought to contribute acidic pKa (pKe1), while no residue was assigned to contribute alkaline pKa (pKe2). In this study, we introduced nine single mutations at the His or Tyr residues in and near the active site. Our results showed that H412A, H485A, Y497A, H578A and H737A retained the activities to hydrolyze collagen and gelatin, while H426A, H492A, H496A and Y568A lacked them. Purification of active variants H412A, H485A, H578A and H737A, along with inactive variants H492A and H496A, were successful. H412A preferred (7-methoxycoumarin-4-yl)acetyl-L-Lys-L-Pro-L-Leu-Gly-L-Leu-[N3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH2 to collagen, while H485A preferred collagen to the peptide, suggesting that His412 and His485 are important for substrate specificity. Purification of the active variant Y497A and inactive variants H426A and Y568A were unsuccessful, suggesting that these three residues were important for stability. Based on the reported crystal structure of clostridial collagenase, Tyr568 of Ghcol is suggested to be involved in catalysis and may be the ionizable residue for pKe2.
Asunto(s)
Colagenasas/metabolismo , Histidina/metabolismo , Tirosina/metabolismo , Vibrionaceae/enzimología , Secuencia de Aminoácidos , Catálisis , Dominio Catalítico , Colagenasas/química , Colagenasas/genética , Colagenasas/aislamiento & purificación , Histidina/química , Histidina/genética , Mutagénesis Sitio-Dirigida/métodos , Mutación , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia , Relación Estructura-Actividad , Especificidad por Sustrato , Tirosina/química , Tirosina/genética , Vibrionaceae/genéticaRESUMEN
Antibodies are widely used for the detection of specific molecules such as peptides, proteins, and chemical compounds. The specificity of an antibody is therefore its most important feature. However, it is very difficult to confirm antibody specificity. Recently, we made a human protein array consisting of 19,712 kinds of recombinant human proteins produced by a wheat cell-free protein production system. Here, we demonstrate a novel protein array technology for antibody validation (CF-PA2Vtech). Full-length human cDNAs were fused to N-terminal FLAG-GST and then synthesized by the wheat cell-free system. To construct a 20 K human protein array, about 10 to 14 kinds of human proteins were mixed and captured in each well by glutathione-conjugated magnetic beads in 12 plates or one plate with 384- or 1536-well format, respectively, using a strong magnetic device. Using this protein array plate, commercially available anti-HA or anti-PD-1 antibody reacted to 13 or three human proteins, respectively. The cross-reactivity of these proteins was also confirmed by immunoblotting. These proteins have a similar epitope, and alanine mutations of these epitope candidates dissolved the reactivity. These results indicated that CF-PA2Vtech is very useful for validation of antibodies against human protein.
Asunto(s)
Anticuerpos/metabolismo , Análisis por Matrices de Proteínas , Proteínas/metabolismo , Antígenos/metabolismo , Células Clonales , Reacciones Cruzadas , Células HEK293 , Humanos , Receptor de Muerte Celular Programada 1/inmunología , Reproducibilidad de los ResultadosRESUMEN
APOBEC3B cytidine deaminase (A3B) catalyzes cytosine into uracil in single-strand DNA and induces C-to-T mutations in genomic DNA of various types of tumors. Accumulation of APOBEC signature mutations is correlated with a worse prognosis for patients with breast cancer or multiple myeloma, suggesting that A3B activity might be a cause of the unfavorable DNA mutations and clonal evolution in these tumors. Phosphorylation of conserved threonine residues of other cytidine deaminases, activation induced deaminase (AID) and APOBEC3G, inhibits their activity. Here we show that protein kinase A (PKA) physically binds to A3B and phosphorylates Thr214. In vitro deaminase assays and foreign DNA editing assays in cells confirm that phosphomimetic A3B mutants, T214D and T214E, completely lose deaminase activity. Molecular dynamics simulation of A3B phosphorylation reveals that Thr214 phosphorylation disrupts binding between the phospho-A3B catalytic core and ssDNA. These mutants still inhibit retroviral infectivity at least partially, and also retain full anti-retrotransposition activity. These results imply that PKA-mediated phosphorylation inhibits A3B mutagenic activity without destructing its innate immune functions. Therefore, PKA activation could reduce further accumulation of mutations in A3B overexpressing tumors.
Asunto(s)
Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Citidina Desaminasa/antagonistas & inhibidores , Citidina Desaminasa/genética , Antígenos de Histocompatibilidad Menor/genética , Mutación , Neoplasias/enzimología , Fosforilación , Dominio Catalítico , Citoplasma/metabolismo , Citosina/química , ADN de Cadena Simple/genética , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Células HeLa , Humanos , Simulación de Dinámica Molecular , Neoplasias/genética , Treonina/químicaRESUMEN
Current anti-hepatitis B virus (HBV) agents have limited effect in curing HBV infection, and thus novel anti-HBV agents with different modes of action are in demand. In this study, we applied AlphaScreen assay to high-throughput screening of small molecules inhibiting the interaction between HBV large surface antigen (LHBs) and the HBV entry receptor, sodium taurocholate cotransporting polypeptide (NTCP). From the chemical screening, we identified that rapamycin, an immunosuppressant, strongly inhibited the LHBs-NTCP interaction. Rapamycin inhibited hepatocyte infection with HBV without significant cytotoxicity. This activity was due to impaired attachment of the LHBs preS1 domain to cell surface. Pretreatment of target cells with rapamycin remarkably reduced their susceptibility to preS1 attachment, while rapamycin pretreatment to preS1 did not affect its attachment activity, suggesting that rapamycin targets the host side. In support of this, a surface plasmon resonance analysis showed a direct interaction of rapamycin with NTCP. Consistently, rapamycin also prevented hepatitis D virus infection, whose entry into cells is also mediated by NTCP. We also identified two rapamycin derivatives, everolimus and temsirolimus, which possessed higher anti-HBV potencies than rapamycin. Thus, this is the first report for application of AlphaScreen technology that monitors a viral envelope-receptor interaction to identify viral entry inhibitors.
Asunto(s)
Antivirales/farmacología , Antígenos de Superficie de la Hepatitis B/metabolismo , Virus de la Hepatitis B/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Simportadores/metabolismo , Células Hep G2 , Hepatitis B/tratamiento farmacológico , Virus de la Hepatitis B/patogenicidad , Hepatitis D/tratamiento farmacológico , Humanos , Terapia Molecular Dirigida/métodos , Precursores de Proteínas/metabolismo , Sirolimus/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Internalización del Virus/efectos de los fármacosRESUMEN
Hepatitis B virus (HBV) constitutes a significant public health burden, and currently available treatment options are not generally curative, necessitating the development of new therapeutics. Here we have applied random non-standard peptide integrated discovery (RaPID) screening to identify small macrocyclic peptide inhibitors of HBV entry that target the cell-surface receptor for HBV, sodium taurocholate cotransporting polypeptide (NTCP). In addition to their anti-HBV activity, these molecules also inhibit cellular entry by the related hepatitis D virus (HDV), and are active against diverse strains of HBV (including clinically relevant nucleos(t)ide analog-resistant and vaccine escaping strains). Importantly, these macrocyclic peptides, in contrast to other NTCP-binding HBV entry inhibitors, exhibited no inhibition of NTCP-mediated bile acid uptake, making them appealing candidates for therapeutic development.
Asunto(s)
Antivirales/farmacología , Virus de la Hepatitis B/efectos de los fármacos , Compuestos Macrocíclicos/farmacología , Péptidos/farmacología , Receptores de Superficie Celular/antagonistas & inhibidores , Antivirales/química , Células Hep G2 , Virus de la Hepatitis B/metabolismo , Humanos , Compuestos Macrocíclicos/química , Pruebas de Sensibilidad Microbiana , Conformación Molecular , Péptidos/química , Receptores de Superficie Celular/metabolismo , Ácido Taurocólico/química , Ácido Taurocólico/farmacologíaRESUMEN
Current anti-hepatitis B virus (HBV) agents including interferons and nucleos(t)ide analogs efficiently suppress HBV infection. However, as it is difficult to eliminate HBV from chronically infected liver, alternative anti-HBV agents targeting a new molecule are urgently needed. In this study, we applied a chemical array to high throughput screening of small molecules that interacted with sodium taurocholate cotransporting polypeptide (NTCP), an entry receptor for HBV. From approximately 30,000 compounds, we identified 74 candidates for NTCP interactants, and five out of these were shown to inhibit HBV infection in cell culture. One of such compound, NPD8716, a coumarin derivative, interacted with NTCP and inhibited HBV infection without causing cytotoxicity. Consistent with its NTCP interaction capacity, this compound was shown to block viral attachment to host hepatocytes. NPD8716 also prevented the infection with hepatitis D virus, but not hepatitis C virus, in agreement with NPD8716 specifically inhibiting NTCP-mediated infection. Analysis of derivative compounds showed that the anti-HBV activity of compounds was apparently correlated with the affinity to NTCP and the capacity to impair NTCP-mediated bile acid uptake. These results are the first to show that the chemical array technology represents a powerful platform to identify novel viral entry inhibitors.
Asunto(s)
Virus de la Hepatitis B/efectos de los fármacos , Transportadores de Anión Orgánico Sodio-Dependiente/agonistas , Simportadores/agonistas , Inhibidores de Proteínas Virales de Fusión/aislamiento & purificación , Inhibidores de Proteínas Virales de Fusión/farmacología , Acoplamiento Viral/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Ácidos y Sales Biliares/metabolismo , Cumarinas/química , Cumarinas/aislamiento & purificación , Cumarinas/farmacología , Células Hep G2 , Hepacivirus/efectos de los fármacos , Virus de la Hepatitis Delta/efectos de los fármacos , Humanos , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Simportadores/metabolismo , Inhibidores de Proteínas Virales de Fusión/químicaRESUMEN
Human APOBEC3B (A3B) deaminates a cytosine into a uracil in single-stranded (ss) DNA, resulting in human cancers. A3B's deamination activity is conferred by its C-terminal domain (CTD). However, little is known about the mechanism by which target sequences are searched and deaminated. Here, we applied a real-time NMR method to elucidate the deamination properties. We found that A3B CTD shows higher activity toward its target sequence in short ssDNA and efficiently deaminates a target sequence located near the center of ssDNA. These properties are quite different from those of well-studied APOBEC3G, which shows higher activity toward its target sequence in long ssDNA and one located close to the 5'-end. The unique properties of the A3B CTD can be rationally interpreted by considering that after nonspecific binding to ssDNA, A3B slides only for a relatively short distance and tends to dissociate from the ssDNA before reaching the target sequence.
Asunto(s)
Citidina Desaminasa/metabolismo , ADN de Cadena Simple/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Secuencia de Bases , Citidina Desaminasa/química , ADN de Cadena Simple/química , Desaminación , Humanos , Imagen por Resonancia Magnética , Antígenos de Histocompatibilidad Menor/química , Modelos Moleculares , Dominios ProteicosRESUMEN
S100A6 is a Ca2+-signal transducer that interacts with numerous proteins and regulates their biochemical functions. Here we identified a centrosomal protein, FOR20 (FOP-related protein of 20 kDa) as a novel S100A6 target by screening protein microarrays carrying 19,676 recombinant GST-fused human proteins. Binding experiments revealed that S100A6 interacts with the N-terminal region (residues 1-30) of FOR20 in a Ca2+-dependent manner in vitro and in living cells. Several S100 proteins including S100A1, A2, A4, A11, B also exhibited Ca2+-dependent interactions with FOR20 as well as S100A6. We found that two distantly related centrosomal proteins, FOP and OFD1, also possess N-terminal regions with a significant sequence similarity to the putative S100A6-binding site (residues 1-30) in FOR20 and are capable of binding to S100A6 in a Ca2+-dependent manner. Taken together, these results may indicate that S100A6 interacts with FOR20 and related centrosomal proteins through a conserved N-terminal domain, suggesting a novel Ca2+-dependent regulation of centrosomal function.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas/química , Proteínas/metabolismo , Proteínas S100/química , Proteínas S100/metabolismo , Animales , Células COS , Células Cultivadas , Centrosoma/química , Centrosoma/metabolismo , Chlorocebus aethiops , Células HeLa , Humanos , Análisis por Matrices de Proteínas , Unión Proteica , Proteína A6 de Unión a Calcio de la Familia S100 , Especificidad por SustratoRESUMEN
Activation of caspases is crucial for the execution of apoptosis. Although the caspase cascade associated with activation of the initiator caspase-8 (CASP8) has been investigated in molecular and biochemical detail, the physiological role of CASP8 is not fully understood. Here, we identified a two-pore domain potassium channel, tandem-pore domain halothane-inhibited K+ channel 1 (THIK-1), as a novel CASP8 substrate. The intracellular region of THIK-1 was cleaved by CASP8 in apoptotic cells. Overexpression of THIK-1, but not its mutant lacking the CASP8-target sequence in the intracellular portion, accelerated cell shrinkage in response to apoptotic stimuli. In contrast, knockdown of endogenous THIK-1 by RNA interference resulted in delayed shrinkage and potassium efflux. Furthermore, a truncated THIK-1 mutant lacking the intracellular region, which mimics the form cleaved by CASP8, led to a decrease of cell volume of cultured cells without apoptotic stimulation and excessively promoted irregular development of Xenopus embryos. Taken together, these results indicate that THIK-1 is involved in the acceleration of cell shrinkage. Thus, we have demonstrated a novel physiological role of CASP8: creating a cascade that advances the cell to the next stage in the apoptotic process.