RESUMO
Recent epidemiological studies have identified interferon regulatory factor 8 (IRF8) as a susceptibility factor for multiple sclerosis (MS). However, how IRF8 influences the neuroinflammatory disease has remained unknown. By studying the role of IRF8 in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, we found that Irf8(-/-) mice are resistant to EAE. Furthermore, expression of IRF8 in antigen-presenting cells (APCs, such as macrophages, dendritic cells, and microglia), but not in T cells, facilitated disease onset and progression through multiple pathways. IRF8 enhanced αvß8 integrin expression in APCs and activated TGF-ß signaling leading to T helper 17 (Th17) cell differentiation. IRF8 induced a cytokine milieu that favored growth and maintenance of Th1 and Th17 cells, by stimulating interleukin-12 (IL-12) and IL-23 production, but inhibiting IL-27 during EAE. Finally, IRF8 activated microglia and exacerbated neuroinflammation. Together, this work provides mechanistic bases by which IRF8 contributes to the pathogenesis of MS.
Assuntos
Inflamação/fisiopatologia , Integrinas/metabolismo , Fatores Reguladores de Interferon/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Células Cultivadas , Células Dendríticas/imunologia , Encefalomielite Autoimune Experimental/fisiopatologia , Citometria de Fluxo , Fatores Reguladores de Interferon/genética , Macrófagos/imunologia , Camundongos , Camundongos Knockout , RNA Mensageiro/genéticaRESUMO
During coevolution with the host, HIV-1 developed the ability to hijack the cellular ubiquitin/proteasome degradation pathway to counteract the antiviral activity of APOBEC3G (A3G), a host cytidine deaminase that can block HIV-1 replication. Abrogation of A3G function involves the HIV-1 Vif protein, which binds A3G and serves as an adapter molecule to recruit A3G to a Cullin5-based E3 ubiquitin ligase complex. Structure-guided mutagenesis of A3G focused on the 14 most surface-exposed Lys residues allowed us to identify four Lys residues (Lys-297, 301, 303, and 334) that are required for Vif-mediated A3G ubiquitination and degradation. Substitution of Arg for these residues confers Vif resistance and restores A3G's antiviral activity in the presence of Vif. In our model, the critical four Lys residues cluster at the C terminus, opposite to the known N-terminal Vif-interaction region in the protein. Thus, spatial constraints imposed by the E3 ligase complex may be an important determinant in Vif-dependent A3G ubiquitination.
Assuntos
Citidina Desaminase/metabolismo , HIV-1/metabolismo , Lisina/metabolismo , Ubiquitinação , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Desaminase APOBEC-3G , Linhagem Celular , Citidina Desaminase/química , Citidina Desaminase/genética , Infecções por HIV/metabolismo , Humanos , Lisina/genética , Ligação Proteica , Estrutura Terciária de Proteína/genéticaRESUMO
Inherently hierarchic nature of proteins makes multiscale computational methods especially useful in the studies of folding and other functional dynamics. With the multiscale strategies, one can achieve improved accuracy and efficiency by coupling the atomistic and the coarse grained simulations. Depending on the problems studied, very different implementation protocols can be used to realize the multiscale idea. Here, we give detailed introductions to the currently used multiscale protocols, together with some recent applications to the protein folding simulations in our group. The advantages and weakness, as well as the application scopes of these multiscale protocols are discussed. The directions for the future developments are also proposed.
Assuntos
Simulação por Computador , Conformação Proteica , Dobramento de ProteínaRESUMO
This is the first study to our knowledge to report a novel mutation in the interferon regulatory factor 8 gene (IRF8G388S ) associated with multiple idiopathic tooth root resorption, a form of periodontal disease. The IRF8G388S variant in the highly conserved C-terminal motif is predicted to alter the protein structure, likely impairing IRF8 function. Functional assays demonstrated that the IRF8G388S mutant promoted osteoclastogenesis and failed to inhibit NFATc1-dependent transcriptional activation when compared with IRF8WT control. Further, similar to subjects with heterozygous IRF8G388S mutation, Irf8+/- mice exhibited increased osteoclast activity in the mandibular alveolar bone surrounding molar teeth. Immunohistochemistry illustrated increased NFATc1 expression in the dentoalveolar region of Irf8-/- and Irf8+/- mice when compared with Irf8+/+ controls. Genomewide analyses revealed that IRF8 constitutively bound to regulatory regions of several thousand genes in osteoclast precursors, and genetic aberration of IRF8 significantly enhanced many osteoclast-specific transcripts. Collectively, this study delineates the critical role of IRF8 in defining osteoclast lineage and osteoclast transcriptional program, which may help in better understanding of various osteoclast-mediated disorders, including periodontal disease. © 2019 American Society for Bone and Mineral Research.
Assuntos
Predisposição Genética para Doença , Fatores Reguladores de Interferon/genética , Mutação/genética , Osteoclastos/metabolismo , Reabsorção da Raiz/genética , Transcrição Gênica , Idoso de 80 Anos ou mais , Animais , Feminino , Humanos , Fatores Reguladores de Interferon/química , Fatores Reguladores de Interferon/deficiência , Interferon gama/farmacologia , Arcada Osseodentária/patologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Linhagem , Reabsorção da Raiz/patologia , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
Inhibitors of endosome acidification or cathepsin proteases attenuated infections mediated by envelope proteins of xenotropic murine leukemia virus-related virus (XMRV) and Ebola virus, as well as ecotropic, amphotropic, polytropic, and xenotropic murine leukemia viruses (MLVs), indicating that infections by these viruses occur through acidic endosomes and require cathepsin proteases in the susceptible cells such as TE671 cells. However, as previously shown, the endosome acidification inhibitors did not inhibit these viral infections in XC cells. It is generally accepted that the ecotropic MLV infection in XC cells occurs at the plasma membrane. Because cathepsin proteases are activated by low pH in acidic endosomes, the acidification inhibitors may inhibit the viral infections by suppressing cathepsin protease activation. The acidification inhibitors attenuated the activities of cathepsin proteases B and L in TE671 cells, but not in XC cells. Processing of cathepsin protease L was suppressed by the acidification inhibitor in NIH3T3 cells, but again not in XC cells. These results indicate that cathepsin proteases are activated without endosome acidification in XC cells. Treatment with an endocytosis inhibitor or knockdown of dynamin 2 expression by siRNAs suppressed MLV infections in all examined cells including XC cells. Furthermore, endosomal cathepsin proteases were required for these viral infections in XC cells as other susceptible cells. These results suggest that infections of XC cells by the MLVs and Ebola virus occur through endosomes and pH-independent cathepsin activation induces pH-independent infection in XC cells.
Assuntos
Ácidos/metabolismo , Ebolavirus/fisiologia , Endossomos/metabolismo , Vírus da Leucemia Murina de Moloney/fisiologia , Animais , Catepsina B/metabolismo , Concanavalina A/farmacologia , Meios de Cultivo Condicionados/farmacologia , Dinaminas/metabolismo , Ebolavirus/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/virologia , Ativação Enzimática/efeitos dos fármacos , Produtos do Gene env/metabolismo , Vetores Genéticos/genética , Doença pelo Vírus Ebola/patologia , Doença pelo Vírus Ebola/virologia , Humanos , Hidrazonas/farmacologia , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Leucemia Experimental/patologia , Leucemia Experimental/virologia , Camundongos , Modelos Biológicos , Vírus da Leucemia Murina de Moloney/efeitos dos fármacos , Células NIH 3T3 , Especificidade de Órgãos/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , RatosRESUMO
During a comparison of the infectivity of mNDK, a CD4-independent human immunodeficiency virus type 1 (HIV-1) strain, to various cell lines, we found that HeLa cells were much less susceptible than 293T and TE671 cells. Hybridoma cells between HeLa and 293T cells were as susceptible as 293T cells, suggesting that cellular factors enhance the mNDK infection in 293T cells. By screening a cDNA expression library in HeLa cells, cystatin C was isolated as an enhancer of the mNDK infection. Because cathepsin B protease, a natural ligand of cystatin C, was upregulated in HeLa cells, we speculated that the high levels of cathepsin B activities were inhibitory to the CD4-independent infection and that cystatin C enhanced the infection by impairing the excessive cathepsin B activity. Consistent with this idea, pretreatment of HeLa cells with 125 µM of CA-074Me, a cathepsin B inhibitor, resulted in an 8-fold enhancement of the mNDK infectivity. Because cathepsin B is activated by low pH in acidic endosomes, we further examined the potential roles of endosomes in the CD4-independent infection. Suppression of endosome acidification or endocytosis by inhibitors or by an Eps15 dominant negative mutant reduced the infectivity of mNDK in which CD4-dependent infections were not significantly impaired. Taken together, these results suggest that endocytosis, endosomal acidification, and cathepsin B activity are involved in the CD4-independent entry of HIV-1.
Assuntos
Catepsina B/metabolismo , Endocitose , Infecções por HIV/patologia , HIV-1/fisiologia , Ácidos , Antígenos CD4/metabolismo , Catepsina B/antagonistas & inibidores , Cloroquina/farmacologia , Cistatina C/metabolismo , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Genes Dominantes , Vetores Genéticos/genética , Células HEK293 , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Células HeLa , Humanos , Hidrazonas/farmacologia , Imunidade Inata/efeitos dos fármacos , Macrolídeos/farmacologia , Inibidores de Proteases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismoRESUMO
Human immunodeficiency virus type 1 (HIV-1) infection is initiated by successive interactions of viral envelope glycoprotein gp120 with two cellular surface proteins, CD4 and chemokine receptor. The two most common chemokine receptors that allow HIV-1 entry are the CCR5 and CXCR4. The CD4 and CCR5 are mainly localized to the particular plasma membrane microdomains, termed raft, which is rich in glycolipids and cholesterol. However, the CXCR4 is localized only partially to the raft region. Although the raft domain is suggested to participate in HIV-1 infection, its role in entry of CXCR4-tropic (X4-tropic) virus is still unclear. Here, we used a combination of CD4-independent infection system and cholesterol-depletion-inducing reagent, methyl-beta-cyclodextrin (MbetaCD), to address the requirement of raft domain in the X4-tropic virus infection. Treatment of CD4-negative, CXCR4-positive human cells with MbetaCD inhibited CD4-independent infection of the X4-tropic strains. This inhibitory effect of the cholesterol depletion was observed even when the CXCR4 was over-expressed on the target cells. Soluble CD4-induced infection was also inhibited by MbetaCD. The MbetaCD had no effect on the levels of cell surface expression of CXCR4. In contrast to these infections, MbetaCD treatment did not inhibit CD4-dependent HIV-1 infection in the wild type CD4-expressing cells. This study and previous reports showing that CD4 mutants localized to non-raft domains function as HIV-1 receptor indicate that CXCR4 clustering in the raft microdomains, rather than CD4, is the key step for the HIV-1 entry.
Assuntos
HIV-1/fisiologia , Microdomínios da Membrana/metabolismo , Receptores CXCR4/metabolismo , Internalização do Vírus , Antimetabólitos/farmacologia , Colesterol/metabolismo , Células HeLa , Humanos , beta-Ciclodextrinas/farmacologiaRESUMO
Recently it has been reported that a cathepsin B inhibitor, CA-074Me, attenuates ecotropic murine leukemia virus (Eco-MLV) infection in NIH3T3 cells, suggesting that cathepsin B is required for the Eco-MLV infection. However, cathepsin B activity was negative or extremely low in NIH3T3 cells. How did CA-074Me attenuate the Eco-MLV infection? The CA-074Me treatment of NIH3T3 cells inhibited cathepsin L activity, and a cathepsin L specific inhibitor, CLIK148, attenuated the Eco-MLV vector infection. These results indicate that the suppression of cathepsin L activity by CA-074Me induces the inhibition of Eco-MLV infection, suggesting that cathepsin L is required for the Eco-MLV infection in NIH3T3 cells. The CA-074Me treatment inhibited the Eco-MLV infection in human cells expressing the exogenous mouse ecotropic receptor and endogenous cathepsins B and L, but the CLIK148 treatment did not, showing that only the cathepsin L suppression by CLIK148 is not enough to prevent the Eco-MLV infection in cells expressing both of cathepsins B and L, and CA-074Me inhibits the Eco-MLV infection by suppressing both of cathepsins B and L. These results suggest that either cathepsin B or L is sufficient for the Eco-MLV infection.
Assuntos
Catepsina L/fisiologia , Vírus da Leucemia Murina/enzimologia , Animais , Sequência de Bases , Catepsina B/antagonistas & inibidores , Catepsina B/genética , Catepsina B/fisiologia , Catepsina L/antagonistas & inibidores , Linhagem Celular , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , Compostos de Epóxi/farmacologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Vírus da Leucemia Murina/efeitos dos fármacos , Vírus da Leucemia Murina/patogenicidade , Leucemia Experimental/etiologia , Leucemia Experimental/prevenção & controle , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Camundongos , Células NIH 3T3 , Piridinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptores Virais/genética , Receptores Virais/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Infecções por Retroviridae/etiologia , Infecções por Retroviridae/prevenção & controle , Infecções Tumorais por Vírus/etiologia , Infecções Tumorais por Vírus/prevenção & controleRESUMO
A Mus dunni tail fibroblast (MDTF) cell line is highly resistant to infection by ecotropic Moloney murine leukemia virus (Mo-MLV). The cationic amino acid transporter type 1 (CAT1) paralogues of murine NIH 3T3 and MDTF cells (mCAT1 and dCAT1, respectively) contain two conserved N-linked glycosylation sites in the third extracellular loop (ECL3, the putative Mo-MLV binding site). Glycosylation of dCAT1 inhibits Mo-MLV infection, but that of mCAT1 does not. Compared with mCAT1, dCAT1 possesses an Ile-to-Val substitution at position 214 and a Gly insertion at position 236 in the ECL3. To determine the residues responsible for the loss of dCAT1 receptor function, mutants of mCAT1 were constructed. The mCAT1/insG receptor (with a Gly residue inserted at mCAT1 position 236) had greatly reduced Mo-MLV receptor function compared with mCAT1. Treatment of mCAT1/insG-expressing cells with tunicamycin, an N-linked glycosylation inhibitor, increased the transduction titre. In addition, the reduced susceptibility to Mo-MLV observed with mCAT1/insG-expressing cells correlated with impaired binding of Mo-MLV. These results show that a single amino acid insertion confers mCAT1 receptor properties on dCAT1 and provide an important insight into the co-evolution of virus-host interactions.
Assuntos
Fibroblastos/metabolismo , Fibroblastos/virologia , Glicoproteínas de Membrana/genética , Vírus da Leucemia Murina de Moloney/fisiologia , Receptores Virais/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Sequência Conservada , Evolução Molecular , Glicosilação , Humanos , Leucemia Experimental/genética , Leucemia Experimental/virologia , Masculino , Glicoproteínas de Membrana/deficiência , Camundongos , Dados de Sequência Molecular , Vírus da Leucemia Murina de Moloney/genética , Mutagênese Sítio-Dirigida , Ratos , Receptores Virais/deficiência , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Ezrin, radixin, and moesin (ERM) proteins supply functional linkage between integral membrane proteins and cytoskeleton in mammalian cells to regulate membrane protein dynamisms and cytoskeleton rearrangement. To assess potential role of the ERM proteins in HIV-1 lifecycle, we examined if suppression of ERM function in human cells expressing HIV-1 infection receptors influences HIV-1 envelope (Env)-mediated HIV-1-vector transduction and cell-cell fusion. Expression of an ezrin dominant negative mutant or knockdown of ezrin, radixin, or moesin with siRNA uniformly decreased transduction titers of HIV-1 vectors having X4-tropic Env. In contrast, transduction titers of R5-tropic Env HIV-1 vectors were decreased only by radixin knockdown: ezrin knockdown had no detectable effects and moesin knockdown rather increased transduction titer. Each of the ERM suppressions had no detectable effects on cell surface expression of CD4, CCR5, and CXCR4 or VSV-Env-mediated HIV-1 vector transductions. Finally, the individual knockdown of ERM mRNAs uniformly decreased efficiency of cell-cell fusion mediated by X4- or R5-tropic Env and HIV-1 infection receptors. These results suggest that (i) the ERM proteins function as positive regulators of infection by X4-tropic HIV-1, (ii) moesin additionally functions as a negative regulator of R5-tropic HIV-1 virus infection at the early step(s) after the membrane fusion, and (iii) receptor protein dynamisms are regulated differently in R5- and X4-tropic HIV-1 infections.
Assuntos
Proteínas do Citoesqueleto/fisiologia , HIV-1/fisiologia , Proteínas de Membrana/fisiologia , Proteínas dos Microfilamentos/fisiologia , Internalização do Vírus , Antígenos CD4/biossíntese , Fusão Celular , Linhagem Celular , Inativação Gênica , Humanos , RNA Interferente Pequeno/genética , Receptores CCR5/biossíntese , Receptores CXCR4/biossíntese , Transdução GenéticaRESUMO
CXCR4 functions as an infection receptor of X4 human immunodeficiency virus type 1 (HIV-1) . CXCR4 is glycosylated at the N-terminal extracellular region, which is important for viral envelope (Env) protein binding. We compared the effects of CXCR4 glycan on the CD4-dependent and -independent infections in human cells by X4 viruses. We found that transduction mediated by Env proteins of CD4-independent HIV-1 strains increased up to 5.5-fold in cells expressing unglycosylated CXCR4, suggesting that the CXCR4 glycan inhibits CD4-independent X4 virus infection. Co-expression of CD4 on the target cell surface or pre-incubation of virus particles with soluble CD4 abrogates the glycan-mediated inhibition of X4 virus infection, suggesting that interaction of Env protein with CD4 counteracts the inhibition. These findings indicate that it will be advantageous for X4 HIV-1 to remain CD4-dependent. A structural model that explains the glycan-mediated inhibition is discussed.
Assuntos
HIV-1/fisiologia , Polissacarídeos/metabolismo , Polissacarídeos/fisiologia , Receptores CXCR4/química , Receptores CXCR4/fisiologia , Ligação Viral , Linhagem Celular , Humanos , Modelos Moleculares , Polissacarídeos/genética , Transdução GenéticaRESUMO
Ecotropic murine leukemia viruses (MLVs) recognize the third extracellular loop of the receptor, cationic amino acid transporter type 1 (CAT1). The CAT1 protein contains two conserved N-linked glycosylation sites in the third extracellular loops of the mouse, rat, and hamster receptors (mCAT1, rCAT1, and hCAT1, respectively). Glycosylation of the rCAT1 and hCAT1 receptors inhibits ecotropic MLV infection of CAT1-expressing cells, but that of the mCAT1 does not afford the cells this protection. As compared to the mCAT1 protein, the rCAT1 and hCAT1 proteins possess three and six amino acid insertions, respectively, in the third extracellular loop. To determine whether these inserted amino acids are associated with ecotropic MLV infection inhibition by glycosylation, several mutants of mCAT1 and rCAT1 receptors were constructed. Of all the mutants generated in the present study, only rCAT1 mutant 1 exhibited detectable protein expression levels. The rCAT1 mutant 1-expressing human NP2 cells were more susceptible to transduction by ecotropic MLV vectors than the wild-type rCAT1-expressing cells. Tunicamycin, an N-glycosylation inhibitor, increased transduction titer in the wild-type rCAT1-expressing cells, but did not do so in the cells expressing either the mCAT1 or rCAT1 mutation 1. An amino acid substitution in the glycosylation site of the wild-type rCAT1 conferred higher infection susceptibility, but that of the rCAT1 mutant 1 did not. As with the wild-type mCAT1 and rCAT1 proteins, the rCAT1 mutants were detected on the cell surface by immunofluorescence microscopy. Tunicamycin treatment did not affect cellular distribution of the rCAT1 mutant 1, wild-type mCAT1 or rCAT1 proteins. These results indicate that the extra amino acids in the rCAT1 (as compared to the mCAT1) are associated with inhibition of ecotropic MLV infection by the rCAT1 glycosylation.