RESUMO
Viral infections in tubular epithelial cells lead to the production of inflammatory cytokines by innate immunity, causing tubulointerstitial nephritis. TLR3 recognizes viral infections and acts via the activation of interferon (IFN)/IFN-stimulated genes (ISGs). This study investigates the role of ISG56, a representative ISG, in TLR3 signaling in cultured human renal proximal tubular epithelial cells (hRPTECs). To this end, hRPTECs were stimulated by a synthetic TLR3 ligand, polyinosinic-polycytidylic acid (poly IC), recombinant human interferon-ß [r(h)IFN-ß] or Japanese encephalitis virus (JEV) infection and assayed for inflammatory cytokine mRNA expression by RT-qPCR, and protein expression via western blotting or ELISA. ISG56 was expressed by poly IC or r(h)IFN-ß and IFN-ß knockdown reduced poly IC-induced expression of ISG56 and CXCL10. Moreover, ISG56 knockdown reduced poly IC- or r(h)IFN-ß-induced expression of CXCL10 at the same time as increasing JEV growth and reducing CXCL10 expression induced by JEV infection. Overall, TLR3 signaling induced IFN-ß-dependent expression of ISG56 and CXCL10. We show that ISG56 possibly plays a critical role in antiviral immunity of hRPTECs by positive regulation of IFN-ß-mediated CXCL10 expression downstream of TLR3.
Assuntos
Quimiocina CXCL10 , Células Epiteliais , Interferon beta , Túbulos Renais Proximais , Receptor 3 Toll-Like , Humanos , Receptor 3 Toll-Like/metabolismo , Receptor 3 Toll-Like/genética , Quimiocina CXCL10/metabolismo , Quimiocina CXCL10/genética , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/citologia , Células Epiteliais/metabolismo , Interferon beta/metabolismo , Interferon beta/genética , Poli I-C/farmacologia , Transdução de Sinais , Células Cultivadas , Imunidade Inata , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Adaptadoras de Transdução de SinalRESUMO
Hepatitis C virus (HCV) is the major cause of chronic hepatitis and hepatocellular carcinoma. Among its structural proteins, the HCV core protein has been implicated in liver disease. Understanding the role of HCV core proteins in viral diseases is crucial to elucidating disease mechanisms and identifying potential drug targets. However, purification challenges hinder the comprehensive elucidation of the structure and biochemical properties of HCV core proteins. In this study, we successfully solubilized bacterially expressed core protein using a high-salt and detergent-containing buffer and bypassed the denaturing-refolding process. Size-exclusion chromatography revealed three distinct peaks in the HCV-infected cell lysate, with the bacterially expressed soluble core protein corresponding to its second peak. Using a combination of affinity, size exclusion, and multi-modal chromatography purification techniques, we achieved a purity of > 95% for the core protein. Analytical ultracentrifugation revealed monomer formation in the solution. Far UV Circular dichroism spectroscopy identified 25.53% alpha helices and 20.26% beta sheets. These findings strongly suggest that the purified core proteins retained one of the native structures observed in HCV-infected cells.
Assuntos
Carcinoma Hepatocelular , Hepatite C , Neoplasias Hepáticas , Humanos , Hepacivirus , Proteínas do Core ViralRESUMO
In the case of the Japanese encephalitis virus (JEV), the envelope protein (E), a major component of viral particles, contains a highly conserved N-linked glycosylation site (E: N154). Glycosylation of the E protein is thought to play an important role in the ability of the virus to attach to target cells during transmission; however, its role in viral particle formation and release remains poorly understood. In this study, we investigated the role of N-glycosylation of flaviviral structural proteins in viral particle formation and secretion by introducing mutations in viral structural proteins or cellular factors involved in glycoprotein transport and processing. The number of secreted subviral particles (SVPs) was significantly reduced in N154A, a glycosylation-null mutant, but increased in D67N, a mutant containing additional glycosylation sites, indicating that the amount of E glycosylation regulates the release of SVPs. SVP secretion was reduced in cells deficient in galactose, sialic acid, and N-acetylglucosamine modifications in the Golgi apparatus; however, these reductions were not significant, suggesting that glycosylation mainly plays a role in pre-Golgi transport. Fluorescent labeling of SVPs using a split green fluorescent protein (GFP) system and time-lapse imaging by retention using selective hooks (RUSH) system revealed that the glycosylation-deficient mutant was arrested before endoplasmic reticulum (ER)- Golgi transport. However, the absence of ERGIC-53 and ERGIC-L, ER-Golgi transport cargo receptors that recognize sugar chains on cargo proteins, does not impair SVP secretion. In contrast, the solubility of the N154A mutant of E or the N15A/T17A mutant of prM in cells was markedly lower than that of the wild type, and proteasome-mediated rapid degradation of these mutants was observed, indicating the significance of glycosylation of both prM and E in proper protein folding and assembly of viral particles in the ER.
Assuntos
Vírus da Encefalite Japonesa (Espécie) , Flavivirus , Glicosilação , Flavivirus/metabolismo , Proteínas do Envelope Viral/metabolismo , Vírus da Encefalite Japonesa (Espécie)/metabolismo , Vírion/metabolismoRESUMO
B19 virus (B19V) is a pathogenic human parvovirus that infects erythroid progenitor cells. Because there are limited in vitro culture systems to propagate this virus, little is known about the molecular mechanisms by which it propagates in cells. In this study, we introduced a HiBiT peptide tag into various loops of VP2 located on the surface of B19V particles and evaluated their ability to form virus-like particles (VLPs). Three independent sites were identified as permissive sites for peptide tag insertion without affecting VLP formation. When the HiBiT tag was introduced into B19V clones (pB19-M20) and transfected into a semipermissive erythroleukemia cell line (UT7/Epo-S1), HiBiT-dependent luciferase activities (HiBiT activities) increased depending on helicase activity of viral NS1. Furthermore, we used a GFP11 tag-split system to visualize VLPs in the GFP1-10-expressing live cells. Time-lapse imaging of green fluorescent protein (GFP)-labeled VLPs revealed that nuclear VLPs were translocated into the cytoplasm only after cell division, suggesting that the breakdown of the nuclear envelope during mitosis contributes to VLP nuclear export. Moreover, HiBiT activities of culture supernatants were dependent on the presence of a detergent, and the released VLPs were associated with extracellular vesicles, as observed under electron microscopy. Treatment with an antimitotic agent (nocodazole) enhanced the release of VLPs. These results suggest that the virions accumulated in the cytoplasm are constitutively released from the cell as membrane-coated vesicles. These properties are likely responsible for viral escape from host immune responses and enhance membrane fusion-mediated transmission. IMPORTANCE Parvovirus particles are expected to be applied as nanoparticles in drug delivery systems. However, little is known about how nuclear-assembled B19 virus (B19V) virions are released from host cells. This study provides evidence of mitosis-dependent nuclear export of B19V and extracellular vesicle-mediated virion release. Moreover, this study provides methods for modifying particle surfaces with various exogenous factors and contributes to the development of fine nanoparticles with novel valuable functions. The pB19-M20 plasmid expressing HiBiT-tagged VP2 is a novel tool to easily quantify VP2 expression. Furthermore, this system can be applied in high-throughput screening of reagents that affect VP2 expression, which might be associated with viral propagation.
Assuntos
Infecções por Parvoviridae , Parvovirus B19 Humano , Humanos , Linhagem Celular , Proteínas de Fluorescência Verde/metabolismo , Parvovirus B19 Humano/metabolismo , Peptídeos/metabolismo , Partículas Artificiais Semelhantes a VírusRESUMO
Parvovirus B19 (B19) belongs to the Erythroparvovirus genus and is known to cause the fifth disease in children. Primary infection of pregnant women is associated with a high risk of hydrops fetalis and stillbirth due to severe fetal anemia. Virus-like particle (VLP) vaccine candidates for B19 have been developed, although none have been approved so far. The B19 phospholipase A2 domain (B19 PLA2), located in the VP1 unique region, is believed to be associated with adverse inflammatory reactions, and previous effective attempts to improve this vaccine modality inserted a mutation to impair the PLA2 activity of VLPs. In this study, we designed VLPs with a deletion mutant of PLA2 (â¿PLA2 B19 VLP), devoid of PLA2 activity, and confirmed their immunogenicity and safe use in vivo. These results were supported by the lack of histological inflammatory reactions at the site of immunization or the production of IL-6 in â¿PLA2 B19 VLP-immunized mice, that were observed in mice immunized with B19 VLPs. CD4+ T cells from mice vaccinated with VLPs and B19-seropositive human samples were not activated by B19 PLA2 stimulation, suggesting that the B19 PLA2 domain does not constitute a major CD4+ T cell epitope. Most importantly, the â¿PLA2 B19 VLPs induced neutralizing antibodies against B19, in levels similar to those found in B19-seropositive human samples, indicating that they could be used as a safe and effective vaccine candidate against B19.
Assuntos
Parvovirus B19 Humano , Vacinas de Partículas Semelhantes a Vírus , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Criança , Epitopos de Linfócito T , Feminino , Humanos , Interleucina-6 , Camundongos , Parvovirus B19 Humano/genética , Fosfolipases A2/genética , GravidezRESUMO
Many positive-stranded RNA viruses encode polyproteins from which viral proteins are generated by processing the polyproteins. This system produces an equal amount of each viral protein, though the required amounts for each protein are not the same. In this study, we found the extra membrane-anchored nonstructural (NS) proteins of Japanese encephalitis virus and dengue virus are rapidly and selectively degraded by the endoplasmic reticulum-associated degradation (ERAD) pathway. Our gene targeting study revealed that ERAD involving Derlin2 and SEL1L, but not Derlin1, is required for the viral genome replication. Derlin2 is predominantly localized in the convoluted membrane (CM) of the viral replication organelle, and viral NS proteins are degraded in the CM. Hence, these results suggest that viral protein homeostasis is regulated by Derlin2-mediated ERAD in the CM, and this process is critical for the propagation of these viruses. IMPORTANCE The results of this study reveal the cellular ERAD system controls the amount of each viral protein in virus-infected cells and that this "viral protein homeostasis" is critical for viral propagation. Furthermore, we clarified that the "convoluted membrane (CM)," which was previously considered a structure with unknown function, serves as a kind of waste dump where viral protein degradation occurs. We also found that the Derlin2/SEL1L/HRD1-specific pathway is involved in this process, whereas the Derlin1-mediated pathway is not. This novel ERAD-mediated fine-tuning system for the stoichiometries of polyprotein-derived viral proteins may represent a common feature among polyprotein-encoding viruses.
Assuntos
Vírus da Dengue/metabolismo , Vírus da Encefalite Japonesa (Espécie)/metabolismo , Degradação Associada com o Retículo Endoplasmático/fisiologia , Proteínas de Membrana/metabolismo , Proteínas/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Vírus da Dengue/crescimento & desenvolvimento , Vírus da Encefalite Japonesa (Espécie)/crescimento & desenvolvimento , Retículo Endoplasmático/metabolismo , Genoma Viral/genética , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Interferência de RNA , RNA Interferente Pequeno/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteína com Valosina/metabolismo , Células Vero , Replicação Viral/fisiologiaRESUMO
In the activation of cell-mediated adaptive immune responses that play major roles in the elimination of virus-infected or tumor cells, it is important that dendritic cells present antigen peptides on major histocompatibility complex (MHC) class I molecules and activate pathogen-specific cytotoxic T lymphocytes (CTL). As exogenous peptide antigens are generally presented on MHC class II but not class I, the development of a method for exogenous antigen delivery that facilitates MHC class I presentation is necessary for a potentially effective vaccine that is expected to provoke cell-mediated adaptive immune responses. Here, we developed extracellular vesicles that incorporate antigenic proteins by utilizing endosomal sorting complexes required for transport (ESCRT)-mediated vesicle formation pathway. Furthermore, we proved that these vesicles could deliver their contents to the cytoplasm of dendritic cells and activate antigen-specific CTLs. These technologies could be applied to the development of novel CTL-inducing peptide vaccines.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Vesículas Extracelulares , Apresentação de Antígeno , Células Dendríticas , Antígenos de Histocompatibilidade Classe I , Antígenos de Histocompatibilidade Classe II , Peptídeos , Linfócitos T CitotóxicosRESUMO
BACKGROUND: We considered the factors of poor adherence to and dropout from sublingual immunotherapy (SLIT) by verifying patient backgrounds 1 year after start of treatment. METHODS: We recruited 38 patients who began SLIT between November 2014 and September 2015. We analyzed their attributes and level of understanding of the treatment, and conducted a self-reported survey on factors behind dropout cases and poor adherence cases. RESULTS: Four patients dropped out 1 year after start of treatment. Three left for reasons related to anxiety about side effects. There were five cases of poor adherence. There was no significant difference between good adherence, poor adherence, and dropout regarding level of understanding of the treatment (p=0.59). In the comparison between good and poor adherence groups, except four dropout patients, the adherence tended to be poor in patients with short duration of disease, smoking patients, and young patients. Continuous rate of SLIT achieved about 90%, suggesting relatively high level of adherence. CONCLUSION: It appears possible that anxiety related to side effects could be a factor affecting dropout from SLIT. There was no significant difference regarding level of understanding of the treatment. The adherence tended to be poor in patients with short duration of disease, smoking patients, and young patients.
Assuntos
Cryptomeria/imunologia , Pólen/imunologia , Rinite Alérgica Sazonal/terapia , Imunoterapia Sublingual , Administração Sublingual , Adulto , Feminino , Humanos , Masculino , Adesão à Medicação , Pessoa de Meia-Idade , Rinite Alérgica Sazonal/imunologia , Imunoterapia Sublingual/efeitos adversos , Fatores de Tempo , Resultado do TratamentoRESUMO
Flavivirus infection induces endoplasmic reticulum (ER) membrane rearrangements to generate a compartment for replication of the viral genome and assembly of viral particles. Using quantitative mass spectrometry, we identified several ESCRT (endosomal sorting complex required for transport) proteins that are recruited to sites of virus replication on the ER. Systematic small interfering RNA (siRNA) screening revealed that release of both dengue virus and Japanese encephalitis virus was dramatically decreased by single depletion of TSG101 or co-depletion of specific combinations of ESCRT-III proteins, resulting in ≥1,000-fold titer reductions. By contrast, release was unaffected by depletion of some core ESCRTs, including VPS4. Reintroduction of ESCRT proteins to siRNA-depleted cells revealed interactions among ESCRT proteins that are crucial for flavivirus budding. Electron-microscopy studies revealed that the CHMP2 and CHMP4 proteins function directly in membrane deformation at the ER. Thus, a unique and specific subset of ESCRT contributes to ER membrane biogenesis during flavivirus infection.
Assuntos
Proteínas de Ligação a DNA/genética , Vírus da Dengue/genética , Vírus da Encefalite Japonesa (Espécie)/genética , Retículo Endoplasmático/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Células Epiteliais/metabolismo , Fatores de Transcrição/genética , Vírion/genética , ATPases Associadas a Diversas Atividades Celulares , Animais , Linhagem Celular , Chlorocebus aethiops , Cricetulus , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Vírus da Dengue/crescimento & desenvolvimento , Vírus da Dengue/metabolismo , Vírus da Encefalite Japonesa (Espécie)/crescimento & desenvolvimento , Vírus da Encefalite Japonesa (Espécie)/metabolismo , Retículo Endoplasmático/virologia , Complexos Endossomais de Distribuição Requeridos para Transporte/antagonistas & inibidores , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Células Epiteliais/virologia , Regulação da Expressão Gênica , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , Células Vero , Vírion/metabolismo , Replicação Viral/genéticaRESUMO
Macroautophagy is a major intracellular degradation system. We previously reported that overexpression of phosphatase-deficient MTMR3, a member of the myotubularin phosphatidylinositol (PI) 3-phosphatase family, leads to induction of autophagy. In this study, we found that MTMR3 interacted with mTORC1, an evolutionarily conserved serine/threonine kinase complex, which regulates cell growth and autophagy in response to environmental stimuli. Furthermore, overexpression of MTMR3 inhibited mTORC1 activity. The N-terminal half of MTMR3, including the PH-G and phosphatase domains, was necessary and sufficient for these effects. Phosphatase-deficient MTMR3 provided more robust suppression of mTORC1 activity than wild-type MTMR3. Furthermore, phosphatase-deficient full length MTMR3 and the phosphatase domain alone were localized to the Golgi. These results suggest a new regulatory mechanism of mTORC1 in association with PI3P.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Complexos Multiproteicos/antagonistas & inibidores , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Substituição de Aminoácidos , Animais , Autofagia/efeitos dos fármacos , Domínio Catalítico , Células Cultivadas , Embrião de Mamíferos/citologia , Ativação Enzimática/efeitos dos fármacos , Deleção de Genes , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteínas Tirosina Fosfatases não Receptoras/antagonistas & inibidores , Proteínas Tirosina Fosfatases não Receptoras/química , Proteínas Tirosina Fosfatases não Receptoras/genética , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteína Regulatória Associada a mTOR , Serina-Treonina Quinases TOR/química , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Homólogo LST8 da Proteína Associada a mTORRESUMO
Stress granules (SGs) are cytoplasmic foci composed of stalled translation preinitiation complexes induced by environmental stress stimuli, including viral infection. Since viral propagation completely depends on the host translational machinery, many viruses have evolved to circumvent the induction of SGs or co-opt SG components. In this study, we found that expression of Japanese encephalitis virus (JEV) core protein inhibits SG formation. Caprin-1 was identified as a binding partner of the core protein by an affinity capture mass spectrometry analysis. Alanine scanning mutagenesis revealed that Lys(97) and Arg(98) in the α-helix of the JEV core protein play a crucial role in the interaction with Caprin-1. In cells infected with a mutant JEV in which Lys(97) and Arg(98) were replaced with alanines in the core protein, the inhibition of SG formation was abrogated, and viral propagation was impaired. Furthermore, the mutant JEV exhibited attenuated virulence in mice. These results suggest that the JEV core protein circumvents translational shutoff by inhibiting SG formation through an interaction with Caprin-1 and facilitates viral propagation in vitro and in vivo.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Grânulos Citoplasmáticos/metabolismo , Vírus da Encefalite Japonesa (Espécie)/patogenicidade , Interações Hospedeiro-Patógeno , Proteínas do Core Viral/metabolismo , Replicação Viral , Substituição de Aminoácidos , Animais , Linhagem Celular , Chlorocebus aethiops , Análise Mutacional de DNA , Humanos , Espectrometria de Massas , Camundongos , Proteínas Mutantes/metabolismo , Ligação Proteica , VirulênciaRESUMO
Hepatitis C virus (HCV) causes chronic liver disease worldwide. HCV Core protein (Core) forms the viral capsid and is crucial for HCV pathogenesis and HCV-induced hepatocellular carcinoma, through its interaction with the host factor proteasome activator PA28γ. Here, using BD-PowerBlot high-throughput Western array, we attempt to further investigate HCV pathogenesis by comparing the protein levels in liver samples from Core-transgenic mice with or without the knockout of PA28γ expression (abbreviated PA28γ(-/-)CoreTG and CoreTG, respectively) against the wild-type (WT). The differentially expressed proteins integrated into the human interactome were shown to participate in compact and well-connected cellular networks. Functional analysis of the interaction networks using a newly developed data warehouse system highlighted cellular pathways associated with vesicular transport, immune system, cellular adhesion, and cell growth and death among others that were prominently influenced by Core and PA28γ in HCV infection. Follow-up assays with in vitro HCV cell culture systems validated VTI1A, a vesicular transport associated factor, which was upregulated in CoreTG but not in PA28γ(-/-)CoreTG, as a novel regulator of HCV release but not replication. Our analysis provided novel insights into the Core-PA28γ interplay in HCV pathogenesis and identified potential targets for better anti-HCV therapy and potentially novel biomarkers of HCV infection.
Assuntos
Autoantígenos/genética , Hepacivirus/fisiologia , Hepatite C/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Proteínas do Core Viral/biossíntese , Animais , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Morte Celular/genética , Linhagem Celular , Técnicas de Inativação de Genes , Hepacivirus/imunologia , Hepatite C/imunologia , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Complexo de Endopeptidases do Proteassoma/deficiência , Mapas de Interação de Proteínas , Proteoma/metabolismo , Proteômica , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/metabolismo , Proteínas Recombinantes/biossíntese , Vesículas Transportadoras/metabolismo , Liberação de Vírus , Replicação ViralRESUMO
Hepatitis C virus (HCV) is one of the most common etiologic agents of chronic liver diseases, including liver cirrhosis and hepatocellular carcinoma. In addition, HCV infection is often associated with extrahepatic manifestations (EHM), including mixed cryoglobulinemia and non-Hodgkin's lymphoma. However, the mechanisms of cell tropism of HCV and HCV-induced EHM remain elusive, because in vitro propagation of HCV has been limited in the combination of cell culture-adapted HCV (HCVcc) and several hepatic cell lines. Recently, a liver-specific microRNA called miR-122 was shown to facilitate the efficient propagation of HCVcc in several hepatic cell lines. In this study, we evaluated the importance of miR-122 on the replication of HCV in nonhepatic cells. Among the nonhepatic cell lines expressing functional HCV entry receptors, Hec1B cells derived from human uterus exhibited a low level of replication of the HCV genome upon infection with HCVcc. Exogenous expression of miR-122 in several cells facilitates efficient viral replication but not production of infectious particles, probably due to the lack of hepatocytic lipid metabolism. Furthermore, expression of mutant miR-122 carrying a substitution in a seed domain was required for efficient replication of mutant HCVcc carrying complementary substitutions in miR-122-binding sites, suggesting that specific interaction between miR-122 and HCV RNA is essential for the enhancement of viral replication. In conclusion, although miR-122 facilitates efficient viral replication in nonhepatic cells, factors other than miR-122, which are most likely specific to hepatocytes, are required for HCV assembly.
Assuntos
Regulação da Expressão Gênica , Hepacivirus/fisiologia , Hepatite C/metabolismo , MicroRNAs/biossíntese , Montagem de Vírus/fisiologia , Genoma Viral/fisiologia , Células HEK293 , Células Hep G2 , Hepatite C/genética , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Fígado/metabolismo , Fígado/virologia , MicroRNAs/genética , Mutação , Especificidade de Órgãos/genética , RNA Viral/genética , RNA Viral/metabolismoRESUMO
Autophagy is a stress response that is upregulated in response to signals such as starvation, growth factor deprivation, endoplasmic reticulum stress, and pathogen infection. Defects in this pathway are the underlying cause of a number of diseases, including metabolic aberrations, infectious diseases, and cancer, which are closely related to hepatic disorders. To date, more than 30 human ATG (autophagy) genes have been reported to regulate autophagosome formation. In this review, we summarize the current understanding of how ATG proteins behave during autophagosome formation in both non-selective and selective autophagy.
RESUMO
Autophagy is a catabolic process that allows cells to digest their cytoplasmic constituents via autophagosome formation and lysosomal degradation. Recently, an autophagy-specific phosphatidylinositol 3-kinase (PI3-kinase) complex, consisting of hVps34, hVps15, Beclin-1, and Atg14L, has been identified in mammalian cells. Atg14L is specific to this autophagy complex and localizes to the endoplasmic reticulum (ER). Knockdown of Atg14L leads to the disappearance of the DFCP1-positive omegasome, which is a membranous structure closely associated with both the autophagosome and the ER. A point mutation in Atg14L resulting in defective ER localization was also defective in the induction of autophagy. The addition of the ER-targeting motif of DFCP1 to this mutant fully complemented the autophagic defect in Atg14L knockout embryonic stem cells. Thus, Atg14L recruits a subset of class III PI3-kinase to the ER, where otherwise phosphatidylinositol 3-phosphate (PI3P) is essentially absent. The Atg14L-dependent appearance of PI3P in the ER makes this organelle the platform for autophagosome formation.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Autofagia/fisiologia , Retículo Endoplasmático/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Relacionadas à Autofagia , Proteína Beclina-1 , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Fagossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de SequênciaRESUMO
Cyanoformates and cyanoformamides are found to add across alkynes by nickel/Lewis acid (LA) cooperative catalysis to give beta-cyano-substituted acrylates and acrylamides, respectively, in highly stereoselective and regioselective manners. The resulting adducts serve as versatile synthetic building blocks through chemoselective transformations of the ester, amide, and cyano groups as demonstrated by the synthesis of typical structures of beta-cyano ester, beta-amino nitrile, gamma-lactam, disubstituted maleic anhydride, and gamma-aminobutyric acid. The related reactions of cyanoformate thioester and benzoyl cyanide, on the other hand, are found to add across alkynes with decarbonylation in the presence of a palladium/LA catalyst.
RESUMO
The ESCRT pathway helps mediate the final abscission step of cytokinesis in mammals and archaea. In mammals, two early acting proteins of the ESCRT pathway, ALIX and TSG101, are recruited to the midbody through direct interactions with the phosphoprotein CEP55. CEP55 resides at the centrosome through most of the cell cycle but then migrates to the midbody at the start of cytokinesis, suggesting that the ESCRT pathway may also have centrosomal links. Here, we have systematically analyzed the requirements for late-acting mammalian ESCRT-III and VPS4 proteins at different stages of mitosis and cell division. We found that depletion of VPS4A, VPS4B, or any of the 11 different human ESCRT-III (CHMP) proteins inhibited abscission. Remarkably, depletion of individual ESCRT-III and VPS4 proteins also altered centrosome and spindle pole numbers, producing multipolar spindles (most ESCRT-III/VPS4 proteins) or monopolar spindles (CHMP2A or CHMP5) and causing defects in chromosome segregation and nuclear morphology. VPS4 proteins concentrated at spindle poles during mitosis and then at midbodies during cytokinesis, implying that these proteins function directly at both sites. We conclude that ESCRT-III/VPS4 proteins function at centrosomes to help regulate their maintenance or proliferation and then at midbodies during abscission, thereby helping ensure the ordered progression through the different stages of cell division.
Assuntos
Adenosina Trifosfatases/metabolismo , Centrossomo/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Fuso Acromático/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/deficiência , Biomarcadores Tumorais/metabolismo , Sobrevivência Celular , Citocinese , DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/deficiência , Células HeLa , Humanos , Imageamento Tridimensional , Mitose , Transporte Proteico , Fatores de Tempo , ATPases Vacuolares Próton-TranslocadorasRESUMO
The newly described yeast endosomal sorting complexes required for transport (ESCRT) protein increased sodium tolerance-1 (Ist1p) binds the late-acting ESCRT proteins Did2p/charged MVB protein (CHMP) 1 and Vps4p and exhibits synthetic vacuolar protein sorting defects when combined with mutations in the Vta1p/LIP5-Vps60p/CHMP5 complex. Here, we report that human IST1 also functions in the ESCRT pathway and is required for efficient abscission during HeLa cell cytokinesis. IST1 binding interactions with VPS4, CHMP1, LIP5, and ESCRT-I were characterized, and the IST1-VPS4 interaction was investigated in detail. Mutational and NMR spectroscopic studies revealed that the IST1 terminus contains two distinct MIT interacting motifs (MIM1 and MIM2) that wrap around and bind in different groves of the MIT helical bundle. IST1, CHMP1, and VPS4 were recruited to the midbodies of dividing cells, and depleting either IST1 or CHMP1 proteins blocked VPS4 recruitment and abscission. In contrast, IST1 depletion did not inhibit human immunodeficiency virus-1 budding. Thus, IST1 and CHMP1 act together to recruit and modulate specific VPS4 activities required during the final stages of cell division.
Assuntos
Citocinese/fisiologia , Proteínas Oncogênicas/fisiologia , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/metabolismo , Animais , Células COS , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Chlorocebus aethiops , Complexos Endossomais de Distribuição Requeridos para Transporte , HIV-1/fisiologia , Células HeLa , Humanos , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/química , Proteínas Oncogênicas/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Técnicas do Sistema de Duplo-Híbrido , ATPases Vacuolares Próton-Translocadoras , Proteínas de Transporte Vesicular/metabolismoRESUMO
The ESCRT pathway mediates membrane remodeling during enveloped virus budding, cytokinesis, and intralumenal endosomal vesicle formation. Late in the pathway, a subset of membrane-associated ESCRT-III proteins display terminal amphipathic "MIM1" helices that bind and recruit VPS4 ATPases via their MIT domains. We now report that VPS4 MIT domains also bind a second, "MIM2" motif found in a different subset of ESCRT-III subunits. The solution structure of the VPS4 MIT-CHMP6 MIM2 complex revealed that MIM2 elements bind in extended conformations along the groove between the first and third helices of the MIT domain. Mutations that block VPS4 MIT-MIM2 interactions inhibit VPS4 recruitment, lysosomal protein targeting, and HIV-1 budding. MIT-MIM2 interactions appear to be common throughout the ESCRT pathway and possibly elsewhere, and we suggest how these interactions could contribute to a mechanism in which VPS4 and ESCRT-III proteins function together to constrict the necks of budding vesicles.