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1.
Nature ; 611(7935): 260-264, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36352135

RESUMO

In most cosmological models, rapid expansion of space marks the first moments of the Universe and leads to the amplification of quantum fluctuations1. The description of subsequent dynamics and related questions in cosmology requires an understanding of the quantum fields of the standard model and dark matter in curved spacetime. Even the reduced problem of a scalar quantum field in an explicitly time-dependent spacetime metric is a theoretical challenge2-5, and thus a quantum field simulator can lead to insights. Here we demonstrate such a quantum field simulator in a two-dimensional Bose-Einstein condensate with a configurable trap6,7 and adjustable interaction strength to implement this model system. We explicitly show the realization of spacetimes with positive and negative spatial curvature by wave-packet propagation and observe particle-pair production in controlled power-law expansion of space, using Sakharov oscillations to extract amplitude and phase information of the produced state. We find quantitative agreement with analytical predictions for different curvatures in time and space. This benchmarks and thereby establishes a quantum field simulator of a new class. In the future, straightforward upgrades offer the possibility to enter unexplored regimes that give further insight into relativistic quantum field dynamics.

2.
Immunity ; 46(3): 333-335, 2017 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-28329695

RESUMO

Caspases have important functions beyond their established role in driving inflammation and apoptosis. In this issue of Immunity, Wang et al. (2017) demonstrate that inflammasome-triggered caspases cleave and inactivate the DNA sensor cGAS, thus restricting the type I interferon response to cytosolic DNA.


Assuntos
Caspases , Nucleotidiltransferases/genética , Citosol/imunologia , Humanos , Inflamassomos , Interferon Tipo I
3.
Nature ; 557(7703): 112-117, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29695863

RESUMO

The linear ubiquitin chain assembly complex (LUBAC) is required for optimal gene activation and prevention of cell death upon activation of immune receptors, including TNFR1 1 . Deficiency in the LUBAC components SHARPIN or HOIP in mice results in severe inflammation in adulthood or embryonic lethality, respectively, owing to deregulation of TNFR1-mediated cell death2-8. In humans, deficiency in the third LUBAC component HOIL-1 causes autoimmunity and inflammatory disease, similar to HOIP deficiency, whereas HOIL-1 deficiency in mice was reported to cause no overt phenotype9-11. Here we show, by creating HOIL-1-deficient mice, that HOIL-1 is as essential for LUBAC function as HOIP, albeit for different reasons: whereas HOIP is the catalytically active component of LUBAC, HOIL-1 is required for LUBAC assembly, stability and optimal retention in the TNFR1 signalling complex, thereby preventing aberrant cell death. Both HOIL-1 and HOIP prevent embryonic lethality at mid-gestation by interfering with aberrant TNFR1-mediated endothelial cell death, which only partially depends on RIPK1 kinase activity. Co-deletion of caspase-8 with RIPK3 or MLKL prevents cell death in Hoil-1-/- (also known as Rbck1-/-) embryos, yet only the combined loss of caspase-8 with MLKL results in viable HOIL-1-deficient mice. Notably, triple-knockout Ripk3-/-Casp8-/-Hoil-1-/- embryos die at late gestation owing to haematopoietic defects that are rescued by co-deletion of RIPK1 but not MLKL. Collectively, these results demonstrate that both HOIP and HOIL-1 are essential LUBAC components and are required for embryogenesis by preventing aberrant cell death. Furthermore, they reveal that when LUBAC and caspase-8 are absent, RIPK3 prevents RIPK1 from inducing embryonic lethality by causing defects in fetal haematopoiesis.


Assuntos
Proteínas de Transporte/metabolismo , Morte Celular , Desenvolvimento Embrionário , Hematopoese , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Caspase 8/genética , Caspase 8/metabolismo , Morte Celular/genética , Perda do Embrião/genética , Desenvolvimento Embrionário/genética , Células Endoteliais/citologia , Feminino , Hematopoese/genética , Camundongos , Camundongos Endogâmicos C57BL , Domínios Proteicos , Proteínas Quinases/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética
4.
Phys Rev Lett ; 131(15): 150201, 2023 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-37897784

RESUMO

We present a general class of entanglement criteria for continuous variable systems. Our criteria are based on the Husimi Q distribution and allow for optimization over the set of all concave functions rendering them extremely general and versatile. We show that several entropic criteria and second moment criteria are obtained as special cases. Our criteria reveal entanglement of families of states undetected by any commonly used criteria and provide clear advantages under typical experimental constraints such as finite detector resolution and measurement statistics.

5.
Eur J Immunol ; 51(6): 1531-1534, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33733474

RESUMO

Immunogenic cancer therapies, including radiation and hypomethylating agents, such as 5-azacytidine, rely on tumor cell-intrinsic activation of the RNA receptor RIG-I for their synergism with immune checkpoint inhibitors. Possible RIG-I ligands are small nuclear RNA (snRNA) and endogenous retroviral elements (ERV) leaking from the nucleus during programmed cell death.


Assuntos
Azacitidina/uso terapêutico , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia/métodos , Melanoma/imunologia , Receptores de Superfície Celular/metabolismo , Animais , Quimiorradioterapia , Modelos Animais de Doenças , Sinergismo Farmacológico , Humanos , Melanoma/terapia , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Superfície Celular/genética , Transdução de Sinais , Resultado do Tratamento
6.
Sensors (Basel) ; 22(4)2022 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-35214521

RESUMO

The success of total hip arthroplasty depends on the experience of the surgeon, and one of the ways the surgeon currently determines the final implant insertion depth is to listen to the change in audible pitch of the hammering sound. We investigated the use of vibration emissions as a novel method for insertion quality assessment. A non-invasive contact microphone-based measurement system for insertion depth estimation, fixation and fracture detection was developed using a simplified in vitro bone/implant (n = 5). A total of 2583 audio recordings were analyzed in vitro to obtain energy spectral density functions. Out of the four main resonant peaks under in vitro conditions, broach insertion depth statistically correlates to increasing 3rd and 4th peak frequencies. Degree of fixation was also observed as higher goodness of fit (0.26-0.78 vs. 0.12-0.51 between two broach sizes, the latter undersized). Finally, however, the moment of fracture could not be predicted. A cadaveric in situ pilot study suggests comparable resonant frequencies in the same order of magnitudes with the bone model. Further understanding of the signal patterns are needed for an early warning system diagnostic system for imminent fractures, bone damage, improving accuracy and quality of future procedures.


Assuntos
Artroplastia de Quadril , Prótese de Quadril , Acústica , Artroplastia de Quadril/métodos , Humanos , Projetos Piloto , Vibração
7.
Eur J Immunol ; 49(3): 504-507, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30585320
8.
Mol Cell Proteomics ; 17(5): 993-1009, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29217617

RESUMO

Coimmunoprecipitation (co-IP) is one of the most frequently used techniques to study protein-protein (PPIs) or protein-nucleic acid interactions (PNIs). However, the presence of coprecipitated contaminants is a well-recognized issue associated with single-step co-IPs. To overcome this limitation, we developed the two-step co-IP (TIP) strategy that enables sequential coimmunoprecipitations of endogenous protein complexes. TIP can be performed with a broad range of mono- and polyclonal antibodies targeting a single protein or different components of a given complex. TIP results in a highly selective enrichment of protein complexes and thus outperforms single-step co-IPs for downstream applications such as mass spectrometry for the identification of PPIs and quantitative PCR for the analysis of PNIs. We benchmarked TIP for the identification of CD95/FAS-interacting proteins in primary human CD4+ T cells, which recapitulated all major known interactors, but also enabled the proteomics discovery of PPM1G and IPO7 as new interaction partners. For its feasibility and high performance, we propose TIP as an advanced tool for the isolation of highly purified protein-protein and protein-nucleic acid complexes under native expression conditions.


Assuntos
Imunoprecipitação/métodos , Complexos Multiproteicos/isolamento & purificação , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Apoptose , Biotinilação , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Imunoprecipitação da Cromatina , Técnicas de Silenciamento de Genes , Humanos , Carioferinas/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Proteína Fosfatase 2C/metabolismo , Proteômica , Receptores Citoplasmáticos e Nucleares/metabolismo , Reprodutibilidade dos Testes , Receptor fas/metabolismo
9.
J Immunol ; 199(7): 2356-2365, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28842469

RESUMO

Maintaining immune tolerance requires the production of Foxp3-expressing regulatory T (Treg) cells in the thymus. Activation of NF-κB transcription factors is critically required for Treg cell development, partly via initiating Foxp3 expression. NF-κB activation is controlled by a negative feedback regulation through the ubiquitin editing enzyme A20, which reduces proinflammatory signaling in myeloid cells and B cells. In naive CD4+ T cells, A20 prevents kinase RIPK3-dependent necroptosis. Using mice deficient for A20 in T lineage cells, we show that thymic and peripheral Treg cell compartments are quantitatively enlarged because of a cell-intrinsic developmental advantage of A20-deficient thymic Treg differentiation. A20-deficient thymic Treg cells exhibit reduced dependence on IL-2 but unchanged rates of proliferation and apoptosis. Activation of the NF-κB transcription factor RelA was enhanced, whereas nuclear translocation of c-Rel was decreased in A20-deficient thymic Treg cells. Furthermore, we found that the increase in Treg cells in T cell-specific A20-deficient mice was already observed in CD4+ single-positive CD25+ GITR+ Foxp3- thymic Treg cell progenitors. Treg cell precursors expressed high levels of the tumor necrosis factor receptor superfamily molecule GITR, whose stimulation is closely linked to thymic Treg cell development. A20-deficient Treg cells efficiently suppressed effector T cell-mediated graft-versus-host disease after allogeneic hematopoietic stem cell transplantation, suggesting normal suppressive function. Holding thymic production of natural Treg cells in check, A20 thus integrates Treg cell activity and increased effector T cell survival into an efficient CD4+ T cell response.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T Reguladores/fisiologia , Timo/citologia , Timo/fisiologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Animais , Apoptose , Diferenciação Celular , Citometria de Fluxo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Proteína Relacionada a TNFR Induzida por Glucocorticoide/genética , Doença Enxerto-Hospedeiro/prevenção & controle , Interleucina-2/imunologia , Ativação Linfocitária , Camundongos , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-rel/genética , Transdução de Sinais , Transplante de Células-Tronco , Timo/imunologia , Fator de Transcrição RelA/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/deficiência
10.
Eur J Immunol ; 47(12): 2153-2162, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28833039

RESUMO

The transfer of regulatory T cells, either freshly isolated, or modified, represents a promising therapeutic approach to dampen misdirected immune responses, like autoimmune diseases, chronic inflammatory syndromes and graft versus host disease. Clinical isolation of highly pure regulatory T cell (Treg) populations is still challenging and labeling reagents can influence their viability and functionality, potentially altering the potency of isolated Treg cell products. Here we show that reversible Fab multimer-based Treg purification can prevent conventional antibody label-induced interferences in vitro and in vivo. Remaining isolation reagents negatively interfere with Treg engraftment efficacy in C57BL/6 wild-type mice due to Fcγ-receptor- as well as IL-2 receptor-mediated mechanisms. Using a preclinical model for acute GvHD, we further show that purified 'label-freed' Tregs are protective at substantially lower cell numbers as compared to conventional nonreversible antibody staining, translating into significantly improved survival of mice treated with minimally manipulated Tregs. These findings have important clinical relevance for future Treg-based cell therapies.


Assuntos
Transferência Adotiva/métodos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/transplante , Animais , Separação Celular/métodos , Células Cultivadas , Feminino , Citometria de Fluxo , Doença Enxerto-Hospedeiro/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Especificidade de Órgãos/imunologia , Reprodutibilidade dos Testes , Fatores de Tempo
11.
Eur J Immunol ; 47(5): 872-879, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28295265

RESUMO

Activation of the C-type lectin receptor Dectin-1 by ß-glucans triggers multiple signals within DCs that result in activation of innate immunity. While these mechanisms can potently prime CD8+ cytotoxic T-cell (CTL) responses without additional adjuvants, the Dectin-1 effector pathways that control CTL induction remain unclear. Here we demonstrate that Dectin-1-induced CTL cross-priming in mice does not require inflammasome activation but strictly depends on the adapter protein Card9 in vitro. In vivo, Dectin-1-mediated Card9 activation after vaccination drives both expansion and activation of Ag-specific CTLs, resulting in long-lasting CTL responses that are sufficient to protect mice from tumor challenge. This Dectin-1-induced antitumor immune response was independent of NK cell function and completely abrogated in Card9-deficient mice. Thus, our results demonstrate that Dectin-1-triggered Card9 signaling but not inflammasome activation can potently cross-prime Ag-specific CTLs, suggesting that this pathway would be a candidate for immunotherapy and vaccine development.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Lectinas Tipo C/metabolismo , Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Proteínas Adaptadoras de Sinalização CARD/deficiência , Proteínas Adaptadoras de Sinalização CARD/genética , Apresentação Cruzada , Imunidade Inata , Inflamassomos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/fisiopatologia , Transdução de Sinais , Vacinação
12.
Eur J Immunol ; 47(11): 1982-1988, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28833031

RESUMO

The NF-κB regulator A20 limits inflammation by providing negative feedback in myeloid cells and B cells. Functional lack of A20 has been linked to several inflammatory and autoimmune diseases. To define how A20 affects the functionality of T effector cells in a highly inflammatory environment, we performed conventional allogeneic hematopoietic stem cell transplantation (allo-HSCT) with A20-deficient CD4+ and CD8+ donor T cells in mice. Severity and mortality of graft-versus-host disease (GVHD) after allo-HSCT was drastically reduced in recipients transplanted with conventional doses of A20-deficient T cells. Consistently, we found that the A20-deficient donor T-cell compartment was strongly diminished at various timepoints after allo-HSCT. However, proportionally more A20-deficient donor T cells produced IFN-γ and systemic inflammation was elevated early after allo-HSCT. Consequently, increasing the dose of transplanted A20-deficient T cells reversed the original phenotype and resulted in enhanced GVHD mortality compared to recipients that received A20+/+ T cells. Still, A20-deficient T cells, activated either through T cell receptor-dependent or -independent mechanisms, were less viable than control A20+/+ T cells, highlighting that A20 balances both, T-cell activation and survival. Thus, our findings suggest that targeting A20 in T cells may allow to modulate T-cell-mediated inflammatory diseases like GVHD.


Assuntos
Doença Enxerto-Hospedeiro/imunologia , Linfócitos T/imunologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/imunologia , Animais , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante Homólogo
13.
Cell Immunol ; 316: 70-76, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28413062

RESUMO

Intact epithelial body surfaces represent physical barriers which protect the organism from invading pathogens and loss of nutrients. Barrier malfunction is closely linked to disorders such as inflammatory bowel disease and graft-versus-host disease. In fact, several pharmacological or radiobiological therapeutic strategies have side effects that affect epithelial surfaces. In this context, assays that accurately assess epithelial barrier integrity in patients and animal models are crucial to create a better understanding of the mechanisms leading to disease or limiting therapeutic approaches due to barrier disruption. Here, we tested the ability of the widely used FITC-dextran intestinal permeability analysis to evaluate loss of intestinal barrier integrity in different murine models of gut mucosal damage and established influx of neutrophil granulocytes into the intestinal lamina propria (LP) as an alternative approach. We demonstrate that the sensitivity and specificity of FITC-dextran intestinal permeability analysis is relatively low: Although it did represent severe forms of mucosal damage due to intensive conditioning therapy (high doses of either total body irradiation (TBI) or chemotherapy) or after conditioning and allogeneic stem cell transplantation, it did not recognize less severe forms of damage as after lower doses of TBI or chemotherapy alone. In addition, discrimination of untreated from irradiated mice by differences in FITC-dextran translocation was not exact. In contrast, influx of neutrophil granulocytes into the intestinal LP, which reflects immune activation due to translocation of microbes and microbial products during intestinal barrier breech, quantitatively correlated with the severity of intestinal barrier damage. It accurately represented both severe and less severe forms of intestinal damage as after high or lower dose TBI or chemotherapy and correctly discriminated treated from untreated animals. Taken together, we demonstrate the limitations of FITC-dextran intestinal permeability analysis and identify intestinal neutrophil influx as a powerful additional tool to measure breakdown of intestinal barrier function.


Assuntos
Enteropatias/imunologia , Infiltração de Neutrófilos , Neutrófilos , Animais , Dextranos/farmacocinética , Modelos Animais de Doenças , Doxorrubicina , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/farmacocinética , Microbioma Gastrointestinal , Enteropatias/patologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Neutrófilos/patologia , Permeabilidade
14.
Immunity ; 28(3): 315-23, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18342006

RESUMO

CpG motifs within phosphorothioate (PS)-modified DNA drive Toll-like receptor 9 (TLR9) activation, but the rules governing recognition of natural phosphodiester (PD) DNA are less understood. Here, we showed that the sugar backbone determined DNA recognition by TLR9. Homopolymeric, base-free PD 2' deoxyribose acted as a basal TLR9 agonist as it bound to and activated TLR9. This effect was enhanced by DNA bases, even short of CpG motifs. In contrast, PS-modified 2' deoxyribose homopolymers acted as TLR9 and TLR7 antagonists. They displayed high affinity to both TLRs and did not activate on their own, but they competitively inhibited ligand-TLR interaction and activation. Although addition of random DNA bases to the PS 2' deoxyribose backbone did not alter these effects, CpG motifs transformed TLR9-inhibitory to robust TLR9-stimulatory activity. Our results identified the PD 2' deoxyribose backbone as an important determinant of TLR9 activation by natural DNA, restrict CpG-motif dependency of TLR9 activation to synthetic PS-modified ligands, and define PS-modified 2' deoxyribose as a prime effector of TLR9 and TLR7 inhibition.


Assuntos
DNA de Cadeia Simples/química , DNA de Cadeia Simples/imunologia , Desoxirribose/imunologia , Receptor Toll-Like 9/imunologia , Animais , Células Dendríticas/imunologia , Endossomos/imunologia , Citometria de Fluxo , Humanos , Masculino , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oligonucleotídeos/química , Oligonucleotídeos/imunologia , Reconhecimento Fisiológico de Modelo , Receptor 7 Toll-Like/imunologia , Receptor 7 Toll-Like/metabolismo
15.
Nature ; 471(7340): 591-6, 2011 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-21455173

RESUMO

Members of the tumour necrosis factor (TNF) receptor superfamily have important functions in immunity and inflammation. Recently linear ubiquitin chains assembled by a complex containing HOIL-1 and HOIP (also known as RBCK1 and RNF31, respectively) were implicated in TNF signalling, yet their relevance in vivo remained uncertain. Here we identify SHARPIN as a third component of the linear ubiquitin chain assembly complex, recruited to the CD40 and TNF receptor signalling complexes together with its other constituents, HOIL-1 and HOIP. Mass spectrometry of TNF signalling complexes revealed RIP1 (also known as RIPK1) and NEMO (also known as IKKγ or IKBKG) to be linearly ubiquitinated. Mutation of the Sharpin gene (Sharpin(cpdm/cpdm)) causes chronic proliferative dermatitis (cpdm) characterized by inflammatory skin lesions and defective lymphoid organogenesis. Gene induction by TNF, CD40 ligand and interleukin-1ß was attenuated in cpdm-derived cells which were rendered sensitive to TNF-induced death. Importantly, Tnf gene deficiency prevented skin lesions in cpdm mice. We conclude that by enabling linear ubiquitination in the TNF receptor signalling complex, SHARPIN interferes with TNF-induced cell death and, thereby, prevents inflammation. Our results provide evidence for the relevance of linear ubiquitination in vivo in preventing inflammation and regulating immune signalling.


Assuntos
Imunidade/imunologia , Inflamação/metabolismo , Transdução de Sinais , Ubiquitinação , Animais , Ligante de CD40/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Linhagem Celular , Humanos , Quinase I-kappa B/metabolismo , Inflamação/patologia , Inflamação/prevenção & controle , Interleucina-1beta/metabolismo , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores do Fator de Necrose Tumoral/deficiência , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Pele/citologia , Pele/imunologia , Pele/metabolismo , Pele/patologia , Fatores de Transcrição , Fator de Necrose Tumoral alfa/deficiência , Fator de Necrose Tumoral alfa/genética , Ubiquitina/química , Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/química , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo
16.
Mol Cell ; 36(5): 831-44, 2009 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-20005846

RESUMO

TNF is a key inflammatory cytokine. Using a modified tandem affinity purification approach, we identified HOIL-1 and HOIP as functional components of the native TNF-R1 signaling complex (TNF-RSC). Together, they were shown to form a linear ubiquitin chain assembly complex (LUBAC) and to ubiquitylate NEMO. We show that LUBAC binds to ubiquitin chains of different linkage types and that its recruitment to the TNF-RSC is impaired in TRADD-, TRAF2-, and cIAP1/2- but not in RIP1- or NEMO-deficient MEFs. Furthermore, the E3 ligase activity of cIAPs, but not TRAF2, is required for HOIL-1 recruitment to the TNF-RSC. LUBAC enhances NEMO interaction with the TNF-RSC, stabilizes this protein complex, and is required for efficient TNF-induced activation of NF-kappaB and JNK, resulting in apoptosis inhibition. Finally, we demonstrate that sustained stability of the TNF-RSC requires LUBAC's enzymatic activity, thereby adding a third form of ubiquitin linkage to the triggering of TNF signaling by the TNF-RSC.


Assuntos
Regulação da Expressão Gênica , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Ubiquitina/metabolismo , Animais , Apoptose , Linhagem Celular , Proteínas Ativadoras de GTPase/genética , Células HeLa , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , NF-kappa B/metabolismo , Transdução de Sinais , Proteína de Domínio de Morte Associada a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/fisiologia , Células U937 , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/fisiologia
19.
Autophagy ; 19(10): 2733-2751, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37418591

RESUMO

Apoptosis is a tightly controlled cell death program executed by proteases, the so-called caspases. It plays an important role in tissue homeostasis and is often dysregulated in cancer. Here, we identified FYCO1, a protein that promotes microtubule plus end-directed transport of autophagic and endosomal vesicles as a molecular interaction partner of activated CASP8 (caspase 8). The absence of FYCO1 sensitized cells to basal and TNFSF10/TRAIL-induced apoptosis by receptor accumulation and stabilization of the Death Inducing Signaling Complex (DISC). Loss of FYCO1 resulted in impaired transport of TNFRSF10B/TRAIL-R2/DR5 (TNF receptor superfamily member 10b) to the lysosomes in TNFSF10/TRAIL-stimulated cells. More in detail, we show that FYCO1 interacted via its C-terminal GOLD domain with the CCZ1-MON1A complex, which is necessary for RAB7A activation and for the fusion of autophagosomal/endosomal vesicles with lysosomes. We demonstrated that FYCO1 is a novel and specific CASP8 substrate. The cleavage at aspartate 1306 resulted in the release of the C-terminal GOLD domain, inactivating FYCO1 function, and allowing for the progression of apoptosis. Furthermore, the lack of FYCO1 resulted in a stronger and prolonged formation of the TNFRSF1A/TNF-R1 signaling complex. Thus, FYCO1 limits the ligand-induced and steady-state signaling of TNFR-superfamily members, providing a control mechanism that fine-tunes both apoptotic and inflammatory answers.Abbreviations: AP: affinity purification; CHX: cycloheximide; co-IP: co-immunoprecipitation; CRISPR: clustered regularly interspaced short palindromic repeats; DISC: death-inducing signaling complex; DR: death receptors; doxy: doxycycline; GEF: guanine nucleotide exchange factor; ind: inducible; KD: knockdown; KO: knockout; MS: mass spectrometry; shRNA: short hairpin RNA; siRNA: small interfering RNA; TIP: two-step co-immunoprecipitation; WB: western blot.


Assuntos
Autofagia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Caspase 8/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Apoptose , Caspases/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Caspase 9/metabolismo
20.
Nat Commun ; 14(1): 4591, 2023 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-37524699

RESUMO

Herpes simplex virus 1 (HSV-1) infection and stress responses disrupt transcription termination by RNA Polymerase II (Pol II). In HSV-1 infection, but not upon salt or heat stress, this is accompanied by a dramatic increase in chromatin accessibility downstream of genes. Here, we show that the HSV-1 immediate-early protein ICP22 is both necessary and sufficient to induce downstream open chromatin regions (dOCRs) when transcription termination is disrupted by the viral ICP27 protein. This is accompanied by a marked ICP22-dependent loss of histones downstream of affected genes consistent with impaired histone repositioning in the wake of Pol II. Efficient knock-down of the ICP22-interacting histone chaperone FACT is not sufficient to induce dOCRs in ΔICP22 infection but increases dOCR induction in wild-type HSV-1 infection. Interestingly, this is accompanied by a marked increase in chromatin accessibility within gene bodies. We propose a model in which allosteric changes in Pol II composition downstream of genes and ICP22-mediated interference with FACT activity explain the differential impairment of histone repositioning downstream of genes in the wake of Pol II in HSV-1 infection.


Assuntos
Herpes Simples , Herpesvirus Humano 1 , Proteínas Imediatamente Precoces , Humanos , Histonas/metabolismo , Herpesvirus Humano 1/genética , Transcrição Gênica , Proteínas Virais/genética , Proteínas Virais/metabolismo , Herpes Simples/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo
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