RESUMO
Hepatitis B virus (HBV) infection is a major global health problem with no established cure. Dedicator of cytokinesis 11 (DOCK11), known as a guanine nucleotide exchange factor (GEF) for Cdc42, is reported to be essential for the maintenance of HBV. However, potential therapeutic strategies targeting DOCK11 have not yet been explored. We have previously developed an in vitro virus method as a more efficient tool for the analysis of proteomics and evolutionary protein engineering. In this study, using the in vitro virus method, we screened and identified a novel antiasialoglycoprotein receptor (ASGR) antibody, ASGR3-10M, and a DOCK11-binding peptide, DCS8-42A, for potential use in HBV infection. We further constructed a fusion protein (10M-D42AN) consisting of ASGR3-10M, DCS8-42A, a fusogenic peptide, and a nuclear localization signal to deliver the peptide inside hepatocytes. We show using immunofluorescence staining that 10M-D42AN was endocytosed into early endosomes and released into the cytoplasm and nucleus. Since DCS8-42A shares homology with activated cdc42-associated kinase 1 (Ack1), which promotes EGFR endocytosis required for HBV infection, we also found that 10M-D42AN inhibited endocytosis of EGFR and Ack1. Furthermore, we show 10M-D42AN suppressed the function of DOCK11 in the host DNA repair system required for covalently closed circular DNA synthesis and suppressed HBV proliferation in mice. In conclusion, this study realizes a novel hepatocyte-specific drug delivery system using an anti-ASGR antibody, a fusogenic peptide, and DOCK11-binding peptide to provide a novel treatment for HBV.
Assuntos
Sistemas de Liberação de Medicamentos , Fatores de Troca do Nucleotídeo Guanina , Vírus da Hepatite B , Hepatite B , Anticorpos de Cadeia Única , Animais , DNA Circular/genética , Receptores ErbB/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hepatite B/tratamento farmacológico , Vírus da Hepatite B/patogenicidade , Vírus da Hepatite B/fisiologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Camundongos , Peptídeos/metabolismo , Anticorpos de Cadeia Única/metabolismo , Replicação Viral/genéticaRESUMO
To date, the prognosis of multiple myeloma (MM) in patients harboring cytogenetic abnormalities (CA) involving t (4; 14) and deletion of chromosome 17 remains poor despite recent advances in drug development that include the use of immunomodulatory drugs (IMiDs) such as lenalidomide for MM. To address this issue, we have developed a novel phenylphthalimide derivative, TC11, that is structurally related to IMiDs. It remains unclear how TC11 induces apoptosis of MM cells with high-risk CA. Here, we show that TC11 does not induce degradation of CRBN's substrates, IKZF1/3 and CK1α, and induces apoptosis of CRBN-silenced MM; this effect was independent of the cereblon (CRBN) pathway, which is involved in the mechanism of action of IMiDs used for the treatment of MM. We also revealed that TC11, in contrast to existing IMiDs, induced degradation of MCL1 and activation of caspase-9. Furthermore, inhibition of CDK1 by CGP74514A prevented TC11-induced MCL1 degradation, caspase-9 activation, and the subsequent apoptotic cell death. We showed that ectopic MCL1 expression rescued apoptosis of MM. These observations suggest that TC11 induces apoptotic death caused by degradation of MCL1 during prolonged mitotic arrest. Therefore, our findings suggest that TC11 is a potential drug candidate for high-risk MM.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Ftalimidas/farmacologia , Antineoplásicos/química , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Fatores Imunológicos/química , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Ftalimidas/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
An oncoprotein MDM2 binds to the extreme N-terminal peptide region of a tumor suppressor protein p53 (p53NTD) and inhibits its anticancer activity. We recently discovered a peptide named MIP which exhibits much higher binding affinity for MDM2 than p53NTD. Experiments showed that the binding free energy (BFE) of MDM2-MIP is lower than that of MDM2-p53NTD by approximately -4 kcal/mol. Here, we develop a theoretical method which is successful in reproducing this quantitative difference and elucidating its physical origins. It enables us to decompose the BFE into a variety of energetic and entropic components, evaluate their relative magnitudes, and identify the physical factors driving or opposing the binding. It should be applicable also to the assessment of differences among ligands in the binding affinity for a particular receptor, which is a central issue in modern chemistry. In the MDM2 case, the higher affinity of MIP is ascribed to a larger gain of translational, configurational entropy of water upon binding. This result is useful to the design of a peptide possessing even higher affinity for MDM2 as a reliable drug against a cancer.
Assuntos
Simulação de Dinâmica Molecular , Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sítios de Ligação , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas c-mdm2/química , Especificidade por Substrato , Termodinâmica , Proteína Supressora de Tumor p53/químicaRESUMO
BACKGROUND: Approximately 50 years have passed since Kawasaki disease (KD) was first reported. The KD nationwide survey began in 1970. Although >360 000 cases have already been reported in Japan, the cause is still unknown. In Japan, the number of patients and incidence rate of KD has continued to increase. It is necessary to examine the trend of the occurrence in the surveillance of KD. METHODS: The nationwide survey of patient incidence in 2015 and 2016 was conducted in 2017, as the 24th nationwide survey of KD. A questionnaire was sent to pediatric departments in hospitals with >100 beds and specialized pediatric hospitals, and was responded to by the attending pediatricians. RESULTS: The total number of patients in 2 years was 31 595, and the sex ratio (male/female) was 1.34. The incidence rate (/100 000 children aged 0-4 years/year) was 330.2 (371.2 in boys, 287.3 in girls) in 2015, and 309.0 (343.2 in boys, 273.2 in girls) in 2016. The number of patients by month peaked in January. The age-specific incidence rate according to sex was highest in children between 9 and 11 months of age, after which the incidence rate gradually decreased with advancing age. CONCLUSIONS: We summarize the most recent nationwide survey of KD and consider the change in the epidemiologic picture.
Assuntos
Síndrome de Linfonodos Mucocutâneos/epidemiologia , Criança , Pré-Escolar , Estudos Epidemiológicos , Feminino , Glucocorticoides/administração & dosagem , Humanos , Imunoglobulinas Intravenosas/administração & dosagem , Incidência , Lactente , Recém-Nascido , Japão/epidemiologia , Masculino , Síndrome de Linfonodos Mucocutâneos/complicações , Síndrome de Linfonodos Mucocutâneos/terapia , Inquéritos e QuestionáriosRESUMO
BACKGROUND: Evidence suggests that seasonal variation in the onset of Kawasaki disease (KD) exists worldwide. Whether a seasonal component to successful i.v. immunoglobulin (IVIG) therapy exists in KD-positive children, however, is unknown. We addressed this question by focusing on patients with primary onset KD who were non-responsive to IVIG treatment, in the large nationwide Japanese KD survey datasets from 2009 to 2016. METHODS: In these datasets, the IVIG therapy non-responders were defined as patients whose fever persisted ≥24 h or recurred ≤24 h after the end of the initial IVIG treatment (dosage, 2,000 mg/kg). Those who successfully responded to this treatment were defined as IVIG responders. The consecutive monthly trend of the proportion of IVIG non-responders was analyzed throughout the study period to investigate seasonal periodicity on Fourier analysis, and the monthly distributions of non-responders and responders were compared. RESULTS: From a total of 113 691 KD-positive patients, 15.7% were IVIG non-responders, and 61% were male. The proportion of non-responders increased across each calendar year with fluctuation, and Fourier analysis indicated seasonal periodicity. The seasonality effect differed between responders and non-responders, with the proportion of responders tending to increase in autumn through winter, while the non-responders showed a decreasing trend in autumn. The seasonality effect tended to differ by sex. CONCLUSIONS: The results indicate that the currently unknown etiological agents of KD might differ between IVIG responders and non-responders. In addition, immune reactivity against such agents possibly differs by sex in the IVIG non-responders.
Assuntos
Imunoglobulinas Intravenosas/uso terapêutico , Fatores Imunológicos/uso terapêutico , Síndrome de Linfonodos Mucocutâneos/tratamento farmacológico , Estações do Ano , Criança , Pré-Escolar , Feminino , Inquéritos Epidemiológicos , Humanos , Lactente , Recém-Nascido , Japão , Masculino , Síndrome de Linfonodos Mucocutâneos/etiologia , Estudos Retrospectivos , Resultado do TratamentoRESUMO
BACKGROUND: The etiology of Kawasaki disease (KD) is unknown. In Japan, the number of patients and incidence rate of KD has increased continuously since its discovery. The aim of this report was to analyze the latest nationwide epidemiological survey of KD in Japan. METHODS: The 23rd nationwide survey of KD was conducted in 2015. To report on all patients diagnosed with KD in 2013 and 2014, a questionnaire was sent to hospitals with ≥100 beds containing pediatric departments, as well as specialized pediatric hospitals. RESULTS: The number of KD patients reported was 15 696 in 2013 and 15 979 in 2014, resulting in an annual incidence rate of 302.5 and 308.0 per 100 000 population aged 0-4 years, respectively. The number of patients and incidence rate of KD in 2014 were the highest ever recorded in Japan. The number of patients diagnosed per month peaked in January, and a gradual increase in summer was also observed. Eight patients died of KD in 2013 and 2014. CONCLUSIONS: The number of patients and incidence rate of KD in Japan continue to increase. Continued surveillance of epidemiological trends of KD is therefore required.
Assuntos
Síndrome de Linfonodos Mucocutâneos/epidemiologia , Adolescente , Criança , Pré-Escolar , Feminino , Inquéritos Epidemiológicos , Humanos , Incidência , Lactente , Recém-Nascido , Japão/epidemiologia , Masculino , Estudos RetrospectivosRESUMO
Despite the development of new drugs for multiple myeloma (MM), the prognosis of MM patients with high-risk cytogenetic abnormalities such as t (4; 14) and del17p remains poor. We reported that a novel phenylphthalimide derivative, TC11, induced apoptosis of MM cells in vitro and in vivo, and TC11 directly bound to α-tubulin and nucleophosmin-1 (NPM1). However, TC11 showed low water solubility and poor pharmacokinetic properties. Here we synthesized a water-soluble TC11-derivative, PEG(E)-TC11, in which HOEtO-TC11 is pegylated with PEG through an ester bond, and we examined its anti-myeloma activity. We observed that PEG(E)-TC11 and its hydrolyzed product, HOEtO-TC11, induced G2/M arrest and the apoptosis of MM cells. Intraperitoneal administration of PEG(E)-TC11 to xenografted mice revealed improved pharmacokinetic properties and significantly delayed tumor growth. TC11 and its derivatives did not bind to cereblon (CRBN), which is a responsible molecule for thalidomide-induced teratogenicity. These results suggest that PEG(E)-TC11 is a good candidate drug for treating high-risk MM.
Assuntos
Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Ftalimidas/administração & dosagem , Ftalimidas/farmacocinética , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Composição de Medicamentos/métodos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Masculino , Taxa de Depuração Metabólica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Mieloma Múltiplo/patologia , Nucleofosmina , Ftalimidas/química , Polietilenoglicóis/química , Fatores de Risco , Resultado do TratamentoRESUMO
BACKGROUND: Since 1987, no study has reported the municipal-level geographical clustering of Kawasaki disease (KD) in Japan. Therefore, the aim of the present study was to identify the temporal and municipal-level geographical clustering of KD. METHODS: The annual incidence rates of KD for each municipality were calculated using nationwide data from 73 758 patients with KD (2007-2012). To determine whether temporal and municipal-level clustering existed, we calculated the correlations of the annual incidence rates for each municipality during the study years, and compared these rates with those of the adjacent municipalities. Spatial scanning analysis was used to identify the geographical clusters for each year, and the incidence rates in those clusters were compared with the rates in the surrounding region. RESULTS: The annual national incidence rate of KD, adjusted for the prefecture-specific response rate, was 322.45 patients per 100 000 children aged 0-4 years. The correlation between the annual incidence rates during 2 consecutive years was significantly positive (coefficients, 0.149-0.428). On spatial scanning analysis, the most likely clusters were in the Tokyo metropolitan area during 2007-2010 and 2012, and in Kumamoto prefecture during 2011. CONCLUSION: Kawasaki disease exhibits temporal and municipal-level clustering.
Assuntos
Síndrome de Linfonodos Mucocutâneos/epidemiologia , Pré-Escolar , Feminino , Humanos , Incidência , Lactente , Recém-Nascido , Japão/epidemiologia , Masculino , Estudos RetrospectivosRESUMO
The nuclear export of proteins is regulated largely through the exportin/CRM1 pathway, which involves the specific recognition of leucine-rich nuclear export signals (NESs) in the cargo proteins, and modulates nuclear-cytoplasmic protein shuttling by antagonizing the nuclear import activity mediated by importins and the nuclear import signal (NLS). Although the prediction of NESs can help to define proteins that undergo regulated nuclear export, current methods of predicting NESs, including computational tools and consensus-sequence-based searches, have limited accuracy, especially in terms of their specificity. We found that each residue within an NES largely contributes independently and additively to the entire nuclear export activity. We created activity-based profiles of all classes of NESs with a comprehensive mutational analysis in mammalian cells. The profiles highlight a number of specific activity-affecting residues not only at the conserved hydrophobic positions but also in the linker and flanking regions. We then developed a computational tool, NESmapper, to predict NESs by using profiles that had been further optimized by training and combining the amino acid properties of the NES-flanking regions. This tool successfully reduced the considerable number of false positives, and the overall prediction accuracy was higher than that of other methods, including NESsential and Wregex. This profile-based prediction strategy is a reliable way to identify functional protein motifs. NESmapper is available at http://sourceforge.net/projects/nesmapper.
Assuntos
Biologia Computacional/métodos , Leucina/química , Sinais de Exportação Nuclear/genética , Análise de Sequência de Proteína/métodos , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/fisiologia , Sequência de Aminoácidos , Animais , Análise Mutacional de DNA , Leucina/genética , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Sinais de Exportação Nuclear/fisiologia , Curva ROC , SoftwareRESUMO
BACKGROUND: Cardiac lesions, such as coronary dilatation, aneurysms, narrowing, myocardial infarction, and valvular lesions, sometimes occur in Kawasaki disease, but most studies have only evaluated cardiac lesions in the later phase of the disease. This study was undertaken to clarify the related factors between cardiac lesions and laboratory data in the initial phase of Kawasaki disease. METHODS: We conducted a cross-sectional study using data for 26 691 patients from the 22nd nationwide survey of Kawasaki disease in Japan, the observation period of which was from January 2011 through December 2012. We excluded patients with recurrent Kawasaki disease and who were more than seven days from the start of symptoms at admission. We analyzed 23 155 cases (13 353 boys; mean age: 923 ± 734 days) with available laboratory data for white blood cell count, platelet count, serum albumin, and C-reactive protein (CRP). RESULTS: Cardiac lesions were detected in 984 cases (656 boys and 328 girls); lesions were classified as coronary dilatation (764 cases), coronary aneurysm (40), giant coronary aneurysm (6), coronary narrowing (3), and valvular lesions (204). The significant related factors of initial coronary dilatation were male sex (odds ratio [OR] 1.73), older age (OR per 100 days increase 1.03), higher platelet count (OR per 10 000 cells/µL increase 1.006), lower albumin (OR per 1 g/dL increase 0.66), and higher CRP (OR per 1 mg/dL increase 1.02). The factors related to coronary aneurysm were higher platelet count (OR 1.01) and lower albumin (OR 0.34). No factors were significantly related to giant coronary aneurysm. The related factors of valvular lesions were age (OR 0.98), and higher CRP (OR 1.05). CONCLUSIONS: Clinicians should consider male sex, older age, higher platelet count, lower albumin levels, and higher CRP levels when assessing risk of cardiac lesions in the initial phase of Kawasaki disease.
Assuntos
Cardiopatias/epidemiologia , Síndrome de Linfonodos Mucocutâneos/sangue , Síndrome de Linfonodos Mucocutâneos/epidemiologia , Proteína C-Reativa/análise , Criança , Pré-Escolar , Estudos Transversais , Feminino , Inquéritos Epidemiológicos , Humanos , Lactente , Japão/epidemiologia , Contagem de Leucócitos/estatística & dados numéricos , Masculino , Contagem de Plaquetas/estatística & dados numéricos , Fatores de Risco , Albumina Sérica/análiseRESUMO
BACKGROUND: The number of patients and incidence rate of Kawasaki disease (KD) are increasing in Japan. We have therefore characterized the latest epidemiological information on KD. METHODS: The 22nd nationwide survey of KD, which targeted patients diagnosed with KD in 2011 and 2012, was conducted in 2013 and included a total of 1983 departments and hospitals. In order to report on all patients with KD during the 2 survey years, we targeted hospitals of 100 beds or more with pediatric departments, or specialized pediatric hospitals. RESULTS: From a total of 1420 hospitals and departments (71.6% response rate), 26,691 KD patients were reported (12,774 in 2011 and 13,917 in 2012; 15,442 males and 11,249 females). The annual incidence rates were 243.1 per 100,000 population aged 0 to 4 years in 2011 and 264.8 in 2012. The number of cases of KD recorded in 2012 was the highest ever reported in Japan. The incidence rate of complete cases was also the highest ever reported in Japan and contributed to the increase in the rate of total cases in recent years. The number of patients diagnosed per month peaked in January, and additional peaks were noted during summer months, although these peaks were lower than those seen in winter. Age-specific incidence rate showed a monomodal distribution with a peak in the latter half of the year in which patients were born. CONCLUSIONS: The number of patients and the incidence rate of KD in Japan continue to increase. A similar trend has also been seen for patients with complete KD.
Assuntos
Síndrome de Linfonodos Mucocutâneos/epidemiologia , Distribuição por Idade , Criança , Pré-Escolar , Feminino , Inquéritos Epidemiológicos , Humanos , Incidência , Lactente , Recém-Nascido , Japão/epidemiologia , Masculino , Estudos Retrospectivos , Distribuição por SexoRESUMO
Overcoming hepatitis B virus (HBV) is a challenging problem because HBV deceives the host immune system. We have found that DENN domain-containing 2A (DENND2A) was essential for HBV maintenance, although its role remains unclear. In this study, we elucidate its function by screening a novel DENND2A-binding peptide, DENP4-3S. DENP4-3S exhibits homology to SAM and SH3 domain-containing protein 1 (SASH1), a scaffold protein involved in Toll-like receptor signaling that promotes proinflammatory cytokine production. We confirmed that DENND2A interacts with SASH1 specifically. Overexpression and knockdown experiments showed that overexpression of DENND2A suppressed the transcriptional activity of NF-κB, and the knockdown of DENND2A promoted it and the production of cytokines and interferons. Here, we constructed a fusion protein (10M-DEN3SN) consisting of an anti-asialoglycoprotein receptor antibody and DENP4-3S to deliver the peptide to hepatocytes specifically. 10M-DEN3SN inhibited the interaction between DENND2A and SASH1, and rescued SASH1 trapped by DENND2A, leading to the upregulation of NF-κB and its downstream signaling. In addition, 10M-DEN3SN suppressed HBV proliferation in PXB chimeric mice. These results with the DENND2A-binding peptide delivered into hepatocytes suggested the involvement of DENND2A, SASH, and NF-κB signaling pathway in the HBV infection and onset of hepatitis. In conclusion, this study indicates that HBV utilizes DENND2A and SASH1 to evade the immune system.IMPORTANCEHepatitis B virus (HBV) is a serious liver infection with no established cure, causing an abnormal host immune response. Here, we identified a novel peptide that interacts with DENN domain-containing 2A (DENND2A), a host factor essential for HBV maintenance. The resulting peptide showed sequence homology, revealing an interaction between DENND2A and the immune system regulator SASH1. This study suggests that DENND2A contributes to HBV infection by suppressing the cellular immune system by inhibiting SASH1. The DENND2A-binding peptide, incorporated into our hepatocyte-specific peptide delivery system, inhibited the DENND2A-SASH1 interaction and promoted the production of cytokines and interferons in cultured hepatocytes. As a consequence, the peptide suppressed HBV proliferation in humanized mice. We report new insights into the role of DENND2A and SASH1 in HBV maintenance and highlight the importance of the immune system.
Assuntos
Vírus da Hepatite B , Hepatite B , Camundongos , Animais , Vírus da Hepatite B/fisiologia , NF-kappa B/metabolismo , Transdução de Sinais , Interferons , Citocinas/metabolismo , Sistema ImunitárioRESUMO
The questions of how proteins first appeared on the primitive earth and how they evolved into functional proteins are fundamental. If we can understand the origins and evolution of proteins, we should be able to create novel functional proteins. Evolutionary protein engineering or directed protein evolution has been used to create artificial proteins with novel functions by repeated mutation, selection, and amplification, mimicking Darwinian evolution in the laboratory. For this purpose, display technology, such as mRNA display, to link genotype with phenotype is extremely important. Here I focus on three hypotheses regarding the origin and evolution of proteins. First, Eigen's GNC hypothesis proposes that the early genetic code began from the directionless codons GNC and GNN, where N denotes U, C, A, or G. Second, Ohno's gene duplication theory proposes that gene duplication produces two functionally redundant, paralogous genes, of which one retains the original function, leaving the second free to evolve adaptively. Third, Gilbert's exon shuffling theory proposes that new genes are formed through shuffling of small segments corresponding to exons. I then review various experimental approaches to evolutionary protein engineering using mRNA display, such as the creation of functional proteins from random sequences with limited sets of amino acids, randomly mutated folded proteins, and block-shuffled sequence proteins, and I discuss the results in relation to these three hypotheses.
Assuntos
Evolução Molecular , Código Genético , RNA Mensageiro/genética , Aminoácidos/genética , Éxons , Duplicação Gênica/genética , Modelos Genéticos , Engenharia de Proteínas , Dobramento de Proteína , Proteínas/genéticaRESUMO
BACKGROUND: The long-term outcomes of Kawasaki disease (KD) are unknown. METHODS: Fifty-two collaborating hospitals collected data on all patients who had received a new definite diagnosis of KD between July 1982 and December 1992. Patients were followed until December 31, 2009 or death. Standardized mortality ratios (SMRs) were calculated based on Japanese vital statistics data. RESULTS: Of the 6576 patients enrolled, 46 (35 males and 11 females) died (SMR: 1.00; 95% CI: 0.73-1.34). Among persons without cardiac sequelae, SMRs were not high after the acute phase of KD (SMR: 0.65; 95% CI: 0.41-0.96). Among persons with cardiac sequelae, 13 males and 1 female died during the observation period (SMR: 1.86; 95% CI: 1.02-3.13). CONCLUSIONS: In this cohort, the mortality rate among Japanese with cardiac sequelae due to KD was significantly higher than that of the general population. In contrast, the rates for males and females without sequelae were not elevated.
Assuntos
Síndrome de Linfonodos Mucocutâneos/mortalidade , Adolescente , Adulto , Feminino , Seguimentos , Cardiopatias/etiologia , Humanos , Japão/epidemiologia , Masculino , Síndrome de Linfonodos Mucocutâneos/complicações , Taxa de Sobrevida , Adulto JovemRESUMO
BACKGROUND & AIMS: Hepatitis B virus (HBV) infection is difficult to cure owing to the persistence of covalently closed circular viral DNA (cccDNA). We performed single-cell transcriptome analysis of newly established HBV-positive and HBV-negative hepatocellular carcinoma cell lines and found that dedicator of cytokinesis 11 (DOCK11) was crucially involved in HBV persistence. However, the roles of DOCK11 in the HBV lifecycle have not been clarified. METHODS: The cccDNA levels were measured by Southern blotting and real-time detection polymerase chain reaction in various hepatocytes including PXB cells by using an HBV-infected model. The retrograde trafficking route of HBV capsid was investigated by super-resolution microscopy, proximity ligation assay, and time-lapse analysis. The downstream molecules of DOCK11 and underlying mechanism were examined by liquid chromatography-tandem mass spectrometry, immunoblotting, and enzyme-linked immunosorbent assay. RESULTS: The cccDNA levels were strongly increased by DOCK11 overexpression and repressed by DOCK11 suppression. Interestingly, DOCK11 functionally associated with retrograde trafficking proteins in the trans-Golgi network (TGN), Arf-GAP with GTPase domain, ankyrin repeat, and pleckstrin homology domain-containing protein 2 (AGAP2), and ADP-ribosylation factor 1 (ARF1), together with HBV capsid, to open an alternative retrograde trafficking route for HBV from early endosomes (EEs) to the TGN and then to the endoplasmic reticulum (ER), thereby avoiding lysosomal degradation. Clinically, DOCK11 levels in liver biopsies from patients with chronic hepatitis B were significantly reduced by entecavir treatment, and this reduction correlated with HBV surface antigen levels. CONCLUSIONS: HBV uses a retrograde trafficking route via EEs-TGN-ER for infection that is facilitated by DOCK11 and serves to maintain cccDNA. Therefore, DOCK11 is a potential therapeutic target to prevent persistent HBV infection.
Assuntos
Hepatite B Crônica , Hepatite B , Humanos , Vírus da Hepatite B/genética , Rede trans-Golgi/metabolismo , Hepatite B/metabolismo , Lisossomos/metabolismoRESUMO
BACKGROUND: HBV infection causes chronic liver disease and leads to the development of HCC. To identify host factors that support the HBV life cycle, we previously established the HC1 cell line that maintains HBV infection and identified host genes required for HBV persistence. METHODS: The present study focused on endothelial lipase (LIPG), which binds to heparan sulfate proteoglycans (HSPGs) in the cell membrane. RESULTS: We found HBV infection was impaired in humanized liver chimeric mouse-derived hepatocytes that were transduced with lentivirus expressing short hairpin RNA against LIPG. Long-term suppression of LIPG combined with entecavir further suppressed HBV replication. LIPG was shown to be involved in HBV attachment to the cell surface by using 2 sodium taurocholate cotransporting peptide (NTCP)-expressing cell lines, and the direct interaction of LIPG and HBV large surface protein was revealed. Heparin and heparinase almost completely suppressed the LIPG-induced increase of HBV attachment, indicating that LIPG accelerated HBV attachment to HSPGs followed by HBV entry through NTCP. Surprisingly, the attachment of a fluorescently labeled NTCP-binding preS1 probe to NTCP-expressing cells was not impaired by heparin, suggesting the HSPG-independent attachment of the preS1 probe to NTCP. Interestingly, attachment of the preS1 probe was severely impaired in LIPG knockdown or knockout cells. Inhibitors of the lipase activity of LIPG similarly impaired the attachment of the preS1 probe to NTCP-expressing cells. CONCLUSIONS: LIPG participates in HBV infection by upregulating HBV attachment to the cell membrane by means of 2 possible mechanisms: increasing HBV attachment to HSPGs or facilitating HSPG-dependent or HSPG-independent HBV attachment to NTCP by its lipase activity.
Assuntos
Hepatite B , Lipase , Animais , Camundongos , Proteoglicanas de Heparan Sulfato/genética , Heparina , Hepatite B/genética , Vírus da Hepatite B , Lipase/genéticaRESUMO
BACKGROUND: Although the number of patients and incidence rate of Kawasaki disease (KD) are increasing in Japan, the most recent epidemiologic features of KD are not known. METHODS: The 21st nationwide survey of KD was conducted in 2011 and included patients treated for the disease in 2009 and 2010. Hospitals specializing in pediatrics, and hospitals with a total of 100 or more beds and a pediatric department, were asked to report all patients with KD during the 2 survey years. RESULTS: A total of 1445 departments and hospitals reported 23,730 KD patients (10,975 in 2009 and 12,755 in 2010): 13,515 boys and 10 215 girls. The annual incidence rates were 206.2 and 239.6 per 100,000 children aged 0 to 4 years in 2009 and 2010, respectively; the 2010 rate was the highest ever reported in Japan. Monthly number of patients peaked during winter to spring months; lower peaks were noted during summer months. However, the seasonal patterns in 2009 and 2010 differed from those of previous years. The age-specific incidence rate had a monomodal distribution, with a peak during the latter half of the year of birth. The prevalences of cardiac lesions during acute KD and cardiac sequelae were higher among infants and older age groups. Despite a decrease in prevalence, the proportion of patients with giant coronary aneurysms-the most severe sequela of KD-did not substantially decrease. CONCLUSIONS: The incidence rate and number of patients with KD continue to increase in Japan.
Assuntos
Síndrome de Linfonodos Mucocutâneos/epidemiologia , Fatores Etários , Criança , Pré-Escolar , Coleta de Dados , Feminino , Cardiopatias/epidemiologia , Cardiopatias/etiologia , Hospitais Pediátricos/estatística & dados numéricos , Humanos , Incidência , Lactente , Recém-Nascido , Japão/epidemiologia , Masculino , Síndrome de Linfonodos Mucocutâneos/complicações , Prevalência , Estudos Retrospectivos , Estações do AnoRESUMO
The cell cycle-dependent nucleocytoplasmic transport of proteins is predominantly regulated by CDK kinase activities; however, it is currently difficult to predict the proteins thus regulated, largely because of the low prediction efficiency of the motifs involved. Here, we report the successful prediction of CDK1-regulated nucleocytoplasmic shuttling proteins using a prediction system for nuclear localization signals (NLSs). By systematic amino acid replacement analyses in budding yeast, we created activity-based profiles for different classes of importin-alpha-dependent NLSs that represent the functional contributions of different amino acids at each position within an NLS class. We then developed a computer program for prediction of the classical importin-alpha/beta pathway-specific NLSs (cNLS Mapper, available at http//nls-mapper.iab.keio.ac.jp/) that calculates NLS activities by using these profiles and an additivity-based motif scoring algorithm. This calculation method achieved significantly higher prediction accuracy in terms of both sensitivity and specificity than did current methods. The search for NLSs that overlap the consensus CDK1 phosphorylation site by using cNLS Mapper identified all previously reported and 5 previously uncharacterized yeast proteins (Yen1, Psy4, Pds1, Msa1, and Dna2) displaying CDK1- and cell cycle-regulated nuclear transport. CDK1 activated or repressed their nuclear import activity, depending on the position of CDK1-phosphorylation sites within NLSs. The application of this strategy to other functional linear motifs should be useful in systematic studies of protein-protein networks.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Sinais de Localização Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Análise de Sequência de Proteína/métodos , Software , Sequência de Aminoácidos , Proteína Quinase CDC2/metabolismo , Ciclo Celular , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/genética , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , alfa Carioferinas/metabolismoRESUMO
Cofactor for acrosome reaction-inducing substance (Co-ARIS) is a steroidal saponin from the starfish Asterias amurensis. Saponins exist in many plants and few animals as self-defensive chemicals, but Co-ARIS has been identified as a cofactor for inducing the acrosome reaction (AR). In A. amurensis, the AR is induced by the cooperative action of egg coat components (ARIS, Co-ARIS, and asterosap); however, the mechanism of action of Co-ARIS is obscure. In this study we elucidated the membrane dynamics involved in the action of Co-ARIS. We found that cholesterol specifically inhibited the Co-ARIS activity for AR induction and detected the binding of labeled compounds with sperm using radioisotope-labeled Co-ARIS. Co-ARIS treatment did not reduce the content of sperm sterols, however, the condition was changed and localization of GM1 ganglioside on the periacrosomal region disappeared. We then developed a caveola-breaking assay, a novel method to detect the effect of chemicals on microdomains of culture cell, and confirmed the disturbance of somatic cell caveolae in the presence of Co-ARIS. Finally, by atomic force microscopy observations and surface plasmon resonance measurements using an artificial membrane, we revealed that Co-ARIS colocalized with GM1 clusters on the microdomains. Through this study, we revealed a capacitation-like event for AR in starfish sperm.
Assuntos
Reação Acrossômica/efeitos dos fármacos , Asterias/química , Asterias/efeitos dos fármacos , Colesterol/farmacologia , Microdomínios da Membrana/química , Microdomínios da Membrana/efeitos dos fármacos , Saponinas/farmacologia , Animais , Sítios de Ligação , Bioensaio , Células CHO , Cavéolas/efeitos dos fármacos , Cavéolas/metabolismo , Colesterol/química , Cricetinae , Cricetulus , Gangliosídeo G(M1)/metabolismo , Cinética , Masculino , Microdomínios da Membrana/metabolismo , Membranas Artificiais , Conformação Molecular , Saponinas/química , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Trítio/metabolismoRESUMO
Bcl-X(L), an antiapoptotic member of the Bcl-2 family, is a mitochondrial protein that inhibits activation of Bax and Bak, which commit the cell to apoptosis, and it therefore represents a potential target for drug discovery. Peptides have potential as therapeutic molecules because they can be designed to engage a larger portion of the target protein with higher specificity. In the present study, we selected 16-mer peptides that interact with Bcl-X(L) from random and degenerate peptide libraries using mRNA display. The selected peptides have sequence similarity with the Bcl-2 family BH3 domains, and one of them has higher affinity (IC(50)=0.9 microM) than Bak BH3 (IC(50)=11.8 microM) for Bcl-X(L) in vitro. We also found that GFP fusions of the selected peptides specifically interact with Bcl-X(L), localize in mitochondria, and induce cell death. Further, a chimeric molecule, in which the BH3 domain of Bak protein was replaced with a selected peptide, retained the ability to bind specifically to Bcl-X(L). These results demonstrate that this selected peptide specifically antagonizes the function of Bcl-X(L) and overcomes the effects of Bcl-X(L) in intact cells. We suggest that mRNA display is a powerful technique to identify peptide inhibitors with high affinity and specificity for disease-related proteins.