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1.
Sci Signal ; 9(456): ra115, 2016 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-27899525

RESUMO

The increased expression of genes induced by type I interferon (IFN) is characteristic of viral infections and systemic lupus erythematosus (SLE). We showed that mitochondrial antiviral signaling (MAVS) protein, which normally forms a complex with retinoic acid gene I (RIG-I)-like helicases during viral infection, was activated by oxidative stress independently of RIG-I helicases. We found that chemically generated oxidative stress stimulated the formation of MAVS oligomers, which led to mitochondrial hyperpolarization and decreased adenosine triphosphate production and spare respiratory capacity, responses that were not observed in similarly treated cells lacking MAVS. Peripheral blood lymphocytes of SLE patients also showed spontaneous MAVS oligomerization that correlated with the increased secretion of type I IFN and mitochondrial oxidative stress. Furthermore, inhibition of mitochondrial reactive oxygen species (ROS) by the mitochondria-targeted antioxidant MitoQ prevented MAVS oligomerization and type I IFN production. ROS-dependent MAVS oligomerization and type I IFN production were reduced in cells expressing the MAVS-C79F variant, which occurs in 30% of sub-Saharan Africans and is linked with reduced type I IFN secretion and milder disease in SLE patients. Patients expressing the MAVS-C79F variant also had reduced amounts of oligomerized MAVS in their plasma compared to healthy controls. Together, our findings suggest that oxidative stress-induced MAVS oligomerization in SLE patients may contribute to the type I IFN signature that is characteristic of this syndrome.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Linfócitos/metabolismo , Mitocôndrias/metabolismo , Multimerização Proteica , Espécies Reativas de Oxigênio/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Substituição de Aminoácidos , Animais , Humanos , Lúpus Eritematoso Sistêmico/genética , Camundongos , Mitocôndrias/genética , Mutação de Sentido Incorreto
2.
PLoS One ; 9(5): e96156, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24816846

RESUMO

Cellular FLIP (c-FLIP) is an enzymatically inactive paralogue of caspase-8 and as such can block death receptor-induced apoptosis. However, independent of death receptors, c-FLIP-Long (c-FLIPL) can heterodimerize with and activate caspase-8. This is critical for promoting the growth and survival of T lymphocytes as well as the regulation of the RIG-I helicase pathway for type I interferon production in response to viral infections. Truncated forms of FLIP also exist in mammalian cells (c-FLIPS) and certain viruses (v-FLIP), which lack the C-terminal domain that activates caspase-8. Thus, the ratio of c-FLIPL to these short forms of FLIP may greatly influence the outcome of an immune response. We examined this model in mice transgenically expressing c-FLIPS in T cells during infection with Coxsackievirus B3 (CVB3). In contrast to our earlier findings of reduced myocarditis and mortality with CVB3 infection of c-FLIPL-transgenic mice, c-FLIPS-transgenic mice were highly sensitive to CVB3 infection as manifested by increased cardiac virus titers, myocarditis score, and mortality compared to wild-type C57BL/6 mice. This observation was paralleled by a reduction in serum levels of IL-10 and IFN-α in CVB3-infected c-FLIPS mice. In vitro infection of c-FLIPS T cells with CVB3 confirmed these results. Furthermore, molecular studies revealed that following infection of cells with CVB3, c-FLIPL associates with mitochondrial antiviral signaling protein (MAVS), increases caspase-8 activity and type I IFN production, and reduces viral replication, whereas c-FLIPS promotes the opposite phenotype.


Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Infecções por Coxsackievirus/metabolismo , Interferon Tipo I/metabolismo , Viremia/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Western Blotting , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Caspase 8/metabolismo , Células Cultivadas , Infecções por Coxsackievirus/genética , Infecções por Coxsackievirus/virologia , Embrião de Mamíferos/citologia , Enterovirus/genética , Enterovirus/fisiologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/virologia , Interações Hospedeiro-Patógeno , Humanos , Interferon Tipo I/sangue , Interferon Tipo I/genética , Interferon-alfa/sangue , Interferon-alfa/genética , Interferon-alfa/metabolismo , Interferon beta/sangue , Interferon beta/genética , Interferon beta/metabolismo , Interleucina-10/sangue , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miocardite/genética , Miocardite/metabolismo , Miocardite/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/metabolismo , Linfócitos T/virologia , Viremia/genética , Viremia/virologia
3.
J Biol Chem ; 289(2): 1183-91, 2014 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-24275659

RESUMO

Caspase-8 is now appreciated to govern both apoptosis following death receptor ligation and cell survival and growth via inhibition of the Ripoptosome. Cells must therefore carefully regulate the high level of caspase-8 activity during apoptosis versus the modest levels observed during cell growth. The caspase-8 paralogue c-FLIP is a good candidate for a molecular rheostat of caspase-8 activity. c-FLIP can inhibit death receptor-mediated apoptosis by competing with caspase-8 for recruitment to FADD. However, full-length c-FLIPL can also heterodimerize with caspase-8 independent of death receptor ligation and activate caspase-8 via an activation loop in the C terminus of c-FLIPL. This triggers cleavage of c-FLIPL at Asp-376 by caspase-8 to produce p43FLIP. The continued function of p43FLIP has, however, not been determined. We demonstrate that acute deletion of endogenous c-FLIP in murine effector T cells results in loss of caspase-8 activity and cell death. The lethality and caspase-8 activity can both be rescued by the transgenic expression of p43FLIP. Furthermore, p43FLIP associates with Raf1, TRAF2, and RIPK1, which augments ERK and NF-κB activation, IL-2 production, and T cell proliferation. Thus, not only is c-FLIP the initiator of caspase-8 activity during T cell activation, it is also an initial caspase-8 substrate, with cleaved p43FLIP serving to both stabilize caspase-8 activity and promote activation of pathways involved with T cell growth.


Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , NF-kappa B/metabolismo , Fragmentos de Peptídeos/metabolismo , Linfócitos T/metabolismo , Animais , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/química , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Caspase 8/genética , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Humanos , Immunoblotting , Interleucina-2/metabolismo , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fragmentos de Peptídeos/genética , Proteínas Proto-Oncogênicas c-raf , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Linfócitos T/citologia , Fator 2 Associado a Receptor de TNF/metabolismo
4.
J Cardiovasc Transl Res ; 7(2): 182-91, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24323874

RESUMO

Under normal conditions, autophagy maintains cardiomyocyte health and integrity through turnover of organelles. During stress, oxygen and nutrient deprivation, or microbial infection, autophagy prolongs cardiomyocyte survival. Sex differences in induction of cell death may to some extent explain the disparity between the sexes in many human diseases. However, sex differences in gene expression, which regulate cell death and autophagy, were so far not taken in consideration to explain the sex bias of viral myocarditis. Coxsackievirus B3 (CVB3)-induced myocarditis is a sex-biased disease, with females being substantially less susceptible than males and sex hormones largely determine this bias. CVB3 was shown to induce and subvert the autophagosome for its optimal viral RNA replication. Gene expression analysis on mouse and human, healthy and CVB3-infected, cardiac samples of both sexes, suggests sex differences in autophagy-related gene expression. This review discusses the aspects of sex bias in autophagy induction in cardiomyocytes.


Assuntos
Autofagia , Infecções por Coxsackievirus/patologia , Infecções por Coxsackievirus/virologia , Enterovirus Humano B/patogenicidade , Miocardite/patologia , Miocardite/virologia , Miócitos Cardíacos/patologia , Miócitos Cardíacos/virologia , Animais , Autofagia/genética , Infecções por Coxsackievirus/genética , Infecções por Coxsackievirus/imunologia , Enterovirus Humano B/imunologia , Feminino , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Masculino , Miocardite/genética , Miocardite/imunologia , Miócitos Cardíacos/imunologia , Medição de Risco , Fatores de Risco , Fatores Sexuais
6.
RNA ; 18(3): 434-48, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22274955

RESUMO

The catalytic activity of the hammerhead ribozyme is limited by its ability to fold into the native tertiary structure. Analysis of folding has been hampered by a lack of assays that can independently monitor the environment of nucleobases throughout the ribozyme-substrate complex in real time. Here, we report the development and application of a new folding assay in which we use pyrrolo-cytosine (pyC) fluorescence to (1) probe active-site formation, (2) examine the ability of peripheral ribozyme domains to support native folding, (3) identify a pH-dependent conformational change within the ribozyme, and (4) explore its influence on the equilibrium between the folded and unfolded core of the hammerhead ribozyme. We conclude that the natural ribozyme folds in two distinct noncooperative steps and the pH-dependent correlation between core folding and activity is linked to formation of the G8-C3 base pair.


Assuntos
Citosina/análogos & derivados , Corantes Fluorescentes/química , Pirróis/química , Dobramento de RNA , RNA Catalítico/química , Pareamento de Bases , Sequência de Bases , Domínio Catalítico/genética , Citosina/química , Concentração de Íons de Hidrogênio , Cinética , Mutação , RNA Catalítico/genética
7.
Future Virol ; 7(12): 1221-1236, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23503762

RESUMO

Picornaviruses are small, nonenveloped, positive-stranded RNA viruses, which cause a wide range of animal and human diseases, based on their distinct tissue and cell type tropisms. Myocarditis, poliomyelitis, hepatitis and the common cold are the most significant human illnesses caused by picornaviruses. The host response to picornaviruses is complex, and the damage to tissues occurs not only from direct viral replication within infected cells. Picornaviruses exhibit an exceptional ability to evade the early innate immune response, resulting in chronic infection and autoimmunity. This review discusses the detailed aspects of the early innate host response to picornaviruses infection mediated by RIG-I-like helicases, their adaptor, mitochondrial ant iviral signaling protein, innate immune-induced apoptosis, and the role of caspase-8 and its regulatory paralog, FLIP, in these processes.

8.
Immunology ; 135(4): 276-86, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22117649

RESUMO

Caspase-8 serves two paradoxical roles in T lymphocytes: it initiates apoptosis following death receptor engagement, and is also indispensible for proliferation following T-cell antigen receptor (TCR) signalling. These opposing processes appear to be controlled by both spatial and quantitative differences in caspase-8 activation. Given differences in the turnover of T-cell subsets, we compared caspase activity and susceptibility to cell death following TCR restimulation in murine CD4(+) and CD8(+) αß T cells and γδ T cells. We observed a spectrum of caspase activity in non-dying effector T cells in which CD4(+) T cells manifested the lowest levels of active caspases whereas γδ T cells manifested the highest levels. Further analysis revealed that most of the difference in T-cell subsets was the result of high levels of active caspase-3 in non-dying effector γδ T cells. Despite this, γδ T cells manifested little spontaneous or CD3 restimulation-induced cell death as the result of confinement of active caspases to the cell membrane. By contrast, CD4(+) T cells were highly sensitive to CD3-induced cell death, associated with the appearance of active caspases in the cytoplasm and cleavage of the caspase substrates Bid and ICAD. Hence, the location and amount of active caspases distinguishes effector T-cell subsets and profoundly influences the fate of the T-cell response.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Caspase 3/metabolismo , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Animais , Apoptose , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Caspase 3/genética , Caspase 8/metabolismo , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Transdução de Sinais
9.
RNA ; 15(1): 44-54, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19029307

RESUMO

The bacterial signal recognition particle (SRP) binds to ribosomes synthesizing inner membrane proteins and, by interaction with the SRP receptor, FtsY, targets them to the translocon at the membrane. Here we probe the conformation of SRP and SRP protein, Ffh, at different stages of targeting by measuring fluorescence resonance energy transfer (FRET) between fluorophores placed at various positions within SRP. Distances derived from FRET indicate that SRP binding to nontranslating ribosomes triggers a global conformational change of SRP that facilitates binding of the SRP receptor, FtsY. Binding of SRP to a signal-anchor sequence exposed on a ribosome-nascent chain complex (RNC) causes a further change of the SRP conformation, involving the flexible part of the Ffh(M) domain, which increases the affinity for FtsY of ribosome-bound SRP up to the affinity exhibited by the isolated NG domain of Ffh. This indicates that in the RNC-SRP complex the Ffh(NG) domain is fully exposed for binding FtsY to form the targeting complex. Binding of FtsY to the RNC-SRP complex results in a limited conformational change of SRP, which may initiate subsequent targeting steps.


Assuntos
Ribossomos/metabolismo , Partícula de Reconhecimento de Sinal/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Conformação Proteica , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Ribossomos/química , Partícula de Reconhecimento de Sinal/metabolismo
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