RESUMO
We recently demonstrated that chemical proteasome inhibition induced inner retinal degeneration, supporting the pivotal roles of the ubiquitin-proteasome system in retinal structural integrity maintenance. In this study, using beclin1-heterozygous (Becn1-Het) mice with autophagic dysfunction, we tested our hypothesis that autophagy could be a compensatory retinal protective mechanism for proteasomal impairment. Despite the reduced number of autophagosome, the ocular tissue morphology and intraocular pressure were normal. Surprisingly, Becn1-Het mice experienced the same extent of retinal degeneration as was observed in wild-type mice, following an intravitreal injection of a chemical proteasome inhibitor. Similarly, these mice equally responded to other chemical insults, including endoplasmic reticulum stress inducer, N-methyl-D-aspartate, and lipopolysaccharide. Interestingly, in cultured neuroblastoma cells, we found that the mammalian target of rapamycin-independent autophagy activators, lithium chloride and rilmenidine, rescued these cells against proteasome inhibition-induced death. These results suggest that Becn1-mediated autophagy is not an effective intrinsic protective mechanism for retinal damage induced by insults, including impaired proteasomal activity; furthermore, autophagic activation beyond normal levels is required to alleviate the cytotoxic effect of proteasomal inhibition. Further studies are underway to delineate the precise roles of different forms of autophagy, and investigate the effects of their activation in rescuing retinal neurons under various pathological conditions.
Assuntos
Autofagia/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Retina/metabolismo , Degeneração Retiniana/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/fisiologia , Humanos , Camundongos , Retina/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
The inflammasome is an intracellular multi-protein complex that orchestrates the release of the pro-inflammatory cytokines IL-1ß and IL-18, and a form of cell death known as pyroptosis. Tyrosine phosphorylation of the inflammasome sensors NLRP3, AIM2, NLRC4, and the adaptor protein, apoptosis-associated speck-like protein (ASC) has previously been demonstrated to be essential in the regulation of the inflammasome. By using the pharmacological protein tyrosine phosphatase (PTPase) inhibitor, phenylarsine oxide (PAO), we have demonstrated that tyrosine dephosphorylation is an essential step for the activation of the NLRP3 and AIM2 inflammasomes in human and murine macrophages. We have also shown that PTPase activity is required for ASC nucleation leading to caspase-1 activation, IL-1ß, and IL-18 processing and release, and cell death. Furthermore, by site-directed mutagenesis of ASC tyrosine residues, we have identified the phosphorylation of tyrosine Y60 and Y137 of ASC as critical for inflammasome assembly and function. Therefore, we report that ASC tyrosine dephosphorylation and phosphorylation are crucial events for inflammasome activation.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas de Ligação a DNA/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fosforilação/fisiologia , Tirosina/metabolismo , Animais , Caspase 1/metabolismo , Linhagem Celular , Citocinas/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Tirosina Fosfatases/metabolismo , Células Th1RESUMO
Interleukin-1ß (IL-1ß) is a major cytokine that initiates and enhances inflammatory responses. Excessive IL-1ß production is a characteristic of most chronic inflammatory diseases, including atherosclerosis, type 2 diabetes, and obesity, which affect a large proportion of the global population. The production of bioactive IL-1ß is mediated by a caspase-1-activating complex known as an 'inflammasome'. The NLRP3 inflammasome has been associated with several human inflammatory and autoimmune diseases and represents a potential therapeutic target for disrupting IL-1ß production. We used molecular modeling guided by molecular dynamics simulations to design α-helical stapled peptides targeting the pyrin domain of the adaptor protein ASC to interrupt the development of its filament, which is crucial for NLRP3 inflammasome formation. The peptides were effectively internalized by human monocytic cells and efficiently suppressed the release of the inflammasome-regulated cytokines IL-1ß and IL-18, following exogenous activation of the NLRP3 inflammasome. The peptides reduced ASC speck formation and caspase-1 processing thereby suppressing pro-IL-1ß processing and release of active IL-1ß. This is the first demonstration of the successful use of stapled peptides designed to target the adaptor protein ASC, and can be extended to other inflammatory pathways to disrupt excessive IL-1ß production.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/química , Peptídeos Penetradores de Células/farmacologia , Inflamassomos/efeitos dos fármacos , Interleucina-1beta/química , Proteína 3 que Contém Domínio de Pirina da Família NLR/química , Sítios de Ligação , Proteínas Adaptadoras de Sinalização CARD/antagonistas & inibidores , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/imunologia , Peptídeos Penetradores de Células/química , Regulação da Expressão Gênica , Humanos , Interações Hidrofóbicas e Hidrofílicas , Inflamassomos/imunologia , Inflamassomos/metabolismo , Interleucina-18/genética , Interleucina-18/imunologia , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Lipopolissacarídeos/farmacologia , Modelos Moleculares , NF-kappa B/genética , NF-kappa B/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Nigericina/farmacologia , Estudo de Prova de Conceito , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Células THP-1 , TermodinâmicaRESUMO
The inflammasome is a multi-protein complex that mediates proteolytic cleavage and release of the pro-inflammatory cytokines IL-1ß and IL-18, and pyroptosis-a form of cell death induced by various pathogenic bacteria. Apoptosis-associated speck-like protein containing a CARD (ASC) has a pivotal role in inflammasome assembly and activation. While ASC function has been primarily implicated in innate immune cells, its contribution to lymphocyte biology is unclear. Here we report that ASC is constitutively expressed in naïve CD4+ T cells together with the inflammasome sensor NLRP3 and caspase-1. When adoptively transferred in immunocompromised Rag1-/- mice, Asc-/- CD4+ T cells exacerbate T-cell-mediated autoimmune colitis. Asc-/- CD4+ T cells exhibit a higher proliferative capacity in vitro than wild-type CD4+ T cells. The increased expansion of Asc-/- CD4+ T cells in vivo correlated with robust TCR-mediated activation, inflammatory activity, and higher metabolic profile toward a highly glycolytic phenotype. These findings identify ASC as a crucial intrinsic regulator of CD4+ T-cell expansion that serves to maintain intestinal homeostasis.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/imunologia , Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Homeostase/imunologia , Intestinos/imunologia , Animais , Apoptose/genética , Apoptose/imunologia , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Caspase 1/genética , Caspase 1/imunologia , Caspase 1/metabolismo , Células Cultivadas , Colite/genética , Colite/imunologia , Colite/metabolismo , Homeostase/genética , Inflamassomos/genética , Inflamassomos/imunologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismoRESUMO
NLRP10 is a nucleotide-binding oligomerization domain-like receptor that functions as an intracellular pattern recognition receptor for microbial products. Here, we generated a Nlrp10-/- mouse to delineate the role of NLRP10 in the host immune response and found that Nlrp10-/- dendritic cells (DCs) elicited sub-optimal IFNγ production by antigen-specific CD4+ T cells compared to wild-type (WT) DCs. In response to T-cell encounter, CD40 ligation or Toll-like receptor 9 stimulation, Nlrp10-/- DCs produced low levels of IL-12, due to a substantial decrease in NF-κB activation. Defective IL-12 production was also evident in vivo and affected IFNγ production by CD4+ T cells. Upon Mycobacterium tuberculosis (Mtb) infection, Nlrp10-/- mice displayed diminished T helper 1-cell responses and increased bacterial growth compared to WT mice. These data indicate that NLRP10-mediated IL-12 production by DCs is critical for IFNγ induction in T cells and contributes to promote the host defense against Mtb.