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1.
J Clin Invest ; 134(11)2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38652550

RESUMO

The immune system can control cancer progression. However, even though some innate immune sensors of cellular stress are expressed intrinsically in epithelial cells, their potential role in cancer aggressiveness and subsequent overall survival in humans is mainly unknown. Here, we show that nucleotide-binding oligomerization domain-like receptor (NLR) family CARD domain-containing 4 (NLRC4) is downregulated in epithelial tumor cells of patients with colorectal cancer (CRC) by using spatial tissue imaging. Strikingly, only the loss of tumor NLRC4, but not stromal NLRC4, was associated with poor immune infiltration (mainly DCs and CD4+ and CD8+ T cells) and accurately predicted progression to metastatic stage IV and decrease in overall survival. By combining multiomics approaches, we show that restoring NLRC4 expression in human CRC cells triggered a broad inflammasome-independent immune reprogramming consisting of type I interferon (IFN) signaling genes and the release of chemokines and myeloid growth factors involved in the tumor infiltration and activation of DCs and T cells. Consistently, such reprogramming in cancer cells was sufficient to directly induce maturation of human DCs toward a Th1 antitumor immune response through IL-12 production in vitro. In multiple human carcinomas (colorectal, lung, and skin), we confirmed that NLRC4 expression in patient tumors was strongly associated with type I IFN genes, immune infiltrates, and high microsatellite instability. Thus, we shed light on the epithelial innate immune sensor NLRC4 as a therapeutic target to promote an efficient antitumor immune response against the aggressiveness of various carcinomas.


Assuntos
Proteínas Adaptadoras de Sinalização CARD , Proteínas de Ligação ao Cálcio , Neoplasias Colorretais , Regulação Neoplásica da Expressão Gênica , Interferon Tipo I , Transdução de Sinais , Feminino , Humanos , Masculino , Proteínas de Ligação ao Cálcio/genética , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Interferon Tipo I/metabolismo , Interferon Tipo I/imunologia , Interferon Tipo I/genética , Linfócitos do Interstício Tumoral/imunologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia
2.
Sci Rep ; 11(1): 15073, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34302001

RESUMO

The estrogen-related receptor alpha (ERRα) is a primary regulator of mitochondrial energy metabolism, function and dynamics, and has been implicated in autophagy and immune regulation. ERRα is abundantly expressed in the intestine and in cells of the immune system. However, its role in inflammatory bowel disease (IBD) remains unknown. Here, we report a protective role of ERRα in the intestine. We found that mice deficient in ERRα were susceptible to experimental colitis, exhibiting increased colon inflammation and tissue damage. This phenotype was mediated by impaired compensatory proliferation of intestinal epithelial cells (IEC) following injury, enhanced IEC apoptosis and necrosis and reduced mucus-producing goblet cell counts. Longitudinal analysis of the microbiota demonstrated that loss of ERRα lead to a reduction in microbiome α-diversity and depletion of healthy gut bacterial constituents. Mechanistically, ERRα mediated its protective effects by acting within the radio-resistant compartment of the intestine. It promoted disease tolerance through transcriptional control of key genes involved in intestinal tissue homeostasis and repair. These findings provide new insights on the role of ERRα in the gut and extends our current knowledge of nuclear receptors implicated in IBD.


Assuntos
Colite/genética , Metabolismo Energético/genética , Doenças Inflamatórias Intestinais/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Receptores de Estrogênio/genética , Animais , Apoptose/genética , Proliferação de Células/genética , Colite/induzido quimicamente , Colite/metabolismo , Colite/patologia , Colo/metabolismo , Colo/patologia , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Homeostase/genética , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/patologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Microbiota/genética , Necrose/genética , Necrose/metabolismo , Necrose/patologia , Receptor ERRalfa Relacionado ao Estrogênio
3.
Sci Rep ; 8(1): 8446, 2018 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-29855523

RESUMO

Activation of the inflammasome pathway is crucial for effective intracellular host defense. The mitochondrial network plays an important role in inflammasome regulation but the mechanisms linking mitochondrial homeostasis to attenuation of inflammasome activation are not fully understood. Here, we report that the Parkinson's disease-associated mitochondrial serine protease HtrA2 restricts the activation of ASC-dependent NLRP3 and AIM2 inflammasomes, in a protease activity-dependent manner. Consistently, disruption of the protease activity of HtrA2 results in exacerbated NLRP3 and AIM2 inflammasome responses in macrophages ex vivo and systemically in vivo. Mechanistically, we show that the HtrA2 protease activity regulates autophagy and controls the magnitude and duration of inflammasome signaling by preventing prolonged accumulation of the inflammasome adaptor ASC. Our findings identify HtrA2 as a non-redundant mitochondrial quality control effector that keeps NLRP3 and AIM2 inflammasomes in check.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Serina Peptidase 2 de Requerimento de Alta Temperatura A/metabolismo , Inflamassomos/metabolismo , Mitocôndrias/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Autofagia , Células da Medula Óssea/citologia , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspase 1/deficiência , Caspase 1/genética , Caspase 1/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Serina Peptidase 2 de Requerimento de Alta Temperatura A/deficiência , Serina Peptidase 2 de Requerimento de Alta Temperatura A/genética , Proteínas Inibidoras de Apoptose/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores
4.
Oncoimmunology ; 6(3): e1287247, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28405519

RESUMO

Breast cancer is the most common cancer in women and the second leading cause of female cancer-related deaths worldwide. Inflammation is an established hallmark of tumorigenesis and an important determinant of tumor outcome and response to therapy. With advances in cancer immunotherapy, there is an urgent need to dissect the contribution of specific immune effectors in cancer development. Here, we genetically investigated the role of the Interleukin-1 (IL-1) receptor 1 (IL-1R1) pathway in breast cancer tumorigenesis and metastasis using the MMTV-PyMT mouse model. Our results indicate that IL-1R1 signaling suppresses mammary tumor cell proliferation early in tumorigenesis and curbs breast cancer outgrowth and pulmonary metastasis. We show that PyMT/Il1r1-/- mice had a higher primary tumor burden and increased mortality rate compared with IL-1R1-sufficient PyMT control mice. This phenotype was independent of the inflammatory caspases-1/-11 but driven by IL-1α, as PyMT/Il1a-/- mice phenocopied PyMT/Il1r1-/- mice. Collectively, our results suggest that IL-1α-mediated IL-1R1 signaling is tumor-suppressive in PyMT-driven breast cancer.

5.
Immunity ; 43(4): 751-63, 2015 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-26384545

RESUMO

The crosstalk between inflammation and tumorigenesis is now clearly established. However, how inflammation is elicited in the metastatic environment and the corresponding contribution of innate immunity pathways in suppressing tumor growth at secondary sites are poorly understood. Here, we show that mice deficient in Nlrp3 inflammasome components had exacerbated liver colorectal cancer metastatic growth, which was mediated by impaired interleukin-18 (IL-18) signaling. Control of tumor growth was independent of differential cancer cell colonization or proliferation, intestinal microbiota effects, or tumoricidal activity by the adaptive immune system. Instead, the inflammasome-IL-18 pathway impacted maturation of hepatic NK cells, surface expression of the death ligand FasL, and capacity to kill FasL-sensitive tumors. Our results define a regulatory signaling circuit within the innate immune system linking inflammasome activation to effective NK-cell-mediated tumor attack required to suppress colorectal cancer growth in the liver.


Assuntos
Adenocarcinoma/secundário , Proteínas de Transporte/fisiologia , Neoplasias Colorretais/patologia , Inflamassomos/fisiologia , Células Matadoras Naturais/imunologia , Neoplasias Hepáticas/secundário , Adenocarcinoma/imunologia , Animais , Proteínas Reguladoras de Apoptose/deficiência , Proteínas de Ligação ao Cálcio/deficiência , Caspase 1/deficiência , Linhagem Celular Tumoral , Neoplasias Colorretais/imunologia , Citotoxicidade Imunológica , Proteínas de Ligação a DNA/deficiência , Proteína Ligante Fas/fisiologia , Microbioma Gastrointestinal , Imunidade Inata , Vigilância Imunológica , Inflamassomos/deficiência , Interleucina-18/fisiologia , Interleucina-1beta/fisiologia , Neoplasias Hepáticas/imunologia , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/fisiologia , Quimera por Radiação , Tolerância a Radiação , Microambiente Tumoral
6.
J Immunol ; 195(5): 2365-73, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26216893

RESUMO

Chronic proliferative dermatitis in mice (cpdm) is a spontaneous multiorgan inflammatory disorder with pathological hallmarks similar to atopic dermatitis and psoriasis in humans. Cpdm mice lack expression of SHANK-associated RH domain-interacting protein, an adaptor of the linear ubiquitin assembly complex, which acts in the NF-κB pathway to promote inflammation and protect from apoptosis and necroptosis. Although skin inflammation in cpdm mice is driven by TNF- and RIPK1-induced cell death, the contribution of initiating innate immunity sensors and additional inflammatory pathways remains poorly characterized. In this article, we show that inflammasome signaling, including the expression and activation of the inflammatory caspase-1 and -11 and IL-1 family cytokines, was highly upregulated in the skin of cpdm mice prior to overt disease onset. Genetic ablation of caspase-1 and -11 from cpdm mice significantly reduced skin inflammation and delayed disease onset, whereas systemic immunological disease persisted. Loss of Nlrp3 also attenuated skin disease, albeit more variably. Strikingly, induction of apoptosis and necroptosis effectors was sharply decreased in the absence of caspase-1 and -11. These results position the inflammasome as an important initiating signal in skin disease pathogenesis and provide novel insights about inflammasome and cell death effector cross-talk in the context of inflammatory diseases.


Assuntos
Proteínas de Transporte/imunologia , Caspase 1/imunologia , Caspases/imunologia , Dermatite/imunologia , Animais , Apoptose/genética , Apoptose/imunologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Caspase 1/genética , Caspase 1/metabolismo , Caspases/genética , Caspases/metabolismo , Caspases Iniciadoras , Dermatite/genética , Dermatite/metabolismo , Immunoblotting , Inflamassomos/genética , Inflamassomos/imunologia , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Proteína 3 que Contém Domínio de Pirina da Família NLR , Necrose/genética , Necrose/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Pele/imunologia , Pele/metabolismo , Pele/patologia
7.
Cell Host Microbe ; 15(1): 23-35, 2014 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-24439895

RESUMO

Cellular inhibitors of apoptosis proteins (cIAPs) are essential regulators of cell death and immunity. The corresponding contributions of IAPs to infectious disease outcomes are relatively unexplored. We find that mice deficient in cIAP2 exhibit increased susceptibility and mortality to influenza A virus infection. The lethality was not due to impaired antiviral immune functions, but rather because of death-receptor-induced programmed necrosis of airway epithelial cells that led to severe bronchiole epithelial degeneration, despite control of viral replication. Pharmacological inhibition of RIPK1 or genetic deletion of Ripk3, both kinases involved in programmed necrosis, rescued cIAP2-deficient mice from influenza-induced lethality. Genetic deletion of the death receptor agonists Fas ligand or TRAIL from the hematopoietic compartment also reversed the susceptibility of cIAP2-deficient mice. Thus, cIAP2-dependent antagonism of RIPK3-mediated programmed necrosis critically protects the host from influenza infection through maintenance of pulmonary tissue homeostasis rather than through pathogen control by the immune system.


Assuntos
Vírus da Influenza A Subtipo H1N1/imunologia , Proteínas Inibidoras de Apoptose/imunologia , Pulmão/imunologia , Necrose/imunologia , Infecções por Orthomyxoviridae/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologia , Mucosa Respiratória/imunologia , Animais , Proteína 3 com Repetições IAP de Baculovírus , Proteína Ligante Fas/deficiência , Proteína Ligante Fas/genética , Proteína Ligante Fas/imunologia , Regulação da Expressão Gênica , Homeostase/imunologia , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Proteínas Inibidoras de Apoptose/deficiência , Proteínas Inibidoras de Apoptose/genética , Pulmão/patologia , Pulmão/virologia , Camundongos , Camundongos Knockout , Necrose/complicações , Necrose/genética , Necrose/mortalidade , Infecções por Orthomyxoviridae/complicações , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/mortalidade , Inibidores de Proteínas Quinases/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Mucosa Respiratória/patologia , Mucosa Respiratória/virologia , Transdução de Sinais , Análise de Sobrevida , Ligante Indutor de Apoptose Relacionado a TNF/deficiência , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Ubiquitina-Proteína Ligases
8.
J Biol Chem ; 289(4): 2230-49, 2014 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-24302731

RESUMO

It is believed that mitochondrial dynamics is coordinated with endosomal traffic rates during cytoskeletal remodeling, but the mechanisms involved are largely unknown. The adenovirus early region 4 ORF4 protein (E4orf4) subverts signaling by Src family kinases (SFK) to perturb cellular morphology, membrane traffic, and organellar dynamics and to trigger cell death. Using E4orf4 as a model, we uncovered a functional connection between mitochondria-shaping proteins and the small GTPase Rab11a, a key regulator of polarized transport via recycling endosomes. We found that E4orf4 induced dramatic changes in the morphology of mitochondria along with their mobilization at the vicinity of a polarized actin network typifying E4orf4 action, in a manner controlled by SFK and Rab11a. Mitochondrial remodeling was associated with increased proximity between Rab11a and mitochondrial membranes, changes in fusion-fission dynamics, and mitochondrial relocalization of the fission factor dynamin-related protein 1 (Drp1), which was regulated by the Rab11a effector protein FIP1/RCP. Knockdown of FIP1/RCP or inhibition of Drp1 markedly impaired mitochondrial remodeling and actin assembly, involving Rab11a-mediated mitochondrial dynamics in E4orf4-induced signaling. A similar mobilization of mitochondria near actin-rich structures was mediated by Rab11 and Drp1 in viral Src-transformed cells and contributed to the biogenesis of podosome rosettes. These findings suggest a role for Rab11a in the trafficking of Drp1 to mitochondria upon SFK activation and unravel a novel functional interplay between Rab11a and mitochondria during reshaping of the cell cytoskeleton, which would facilitate mitochondria redistribution near energy-requiring actin-rich structures.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo , Citoesqueleto de Actina/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenoviridae/genética , Adenoviridae/metabolismo , Linhagem Celular Transformada , Dinaminas , GTP Fosfo-Hidrolases/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Transdução de Sinais/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas rab de Ligação ao GTP/genética , Quinases da Família src/genética
9.
Cell Signal ; 22(11): 1604-14, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20417707

RESUMO

Evidence has accumulated that there are different modes of regulated cell death, which share overlapping signaling pathways. Cytoskeletal-dependent inter-organellar communication as a result of protein and lipid trafficking in and out of organelles has emerged as a common, key issue in the regulation of cell death modalities. The movement of proteins and lipids between cell compartments is believed to relay death signals in part through modifications of organelles dynamics. Little is known, however, regarding how trafficking is integrated within stress signaling pathways directing organelle-specific remodeling events. In this review, we discuss emerging evidence supporting a role for regulated changes in actin dynamics and intracellular membrane flow. Based on recent findings using the adenovirus E4orf4 death factor as a probing tool to tackle the mechanistic underpinnings that control alternative modes of cell death, we propose the existence of multifunctional platforms at the endosome-Golgi interface regulated by SFK-signaling. These endosomal platforms could be mobilized during cell activation processes to reorganize cellular membranes and promote inter-organelle signaling.


Assuntos
Actinas/metabolismo , Adenoviridae/metabolismo , Proteínas Virais/metabolismo , Quinases da Família src/metabolismo , Apoptose , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Mitocôndrias/metabolismo , Transdução de Sinais
10.
Mol Biol Cell ; 20(18): 4091-106, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19641023

RESUMO

Actin dynamics and membrane trafficking influence cell commitment to programmed cell death through largely undefined mechanisms. To investigate how actin and recycling endosome (RE) trafficking can engage death signaling, we studied the death program induced by the adenovirus early region 4 open reading frame 4 (E4orf4) protein as a model. We found that in the early stages of E4orf4 expression, Src-family kinases (SFKs), Cdc42, and actin perturbed the organization of the endocytic recycling compartment and promoted the transport of REs to the Golgi apparatus, while inhibiting recycling of protein cargos to the plasma membrane. The resulting changes in Golgi membrane dynamics that relied on actin-regulated Rab11a membrane trafficking triggered scattering of Golgi membranes and contributed to the progression of cell death. A similar mobilization of RE traffic mediated by SFKs, Cdc42 and Rab11a also contributed to Golgi fragmentation and to cell death progression in response to staurosporine, in a caspase-independent manner. Collectively, these novel findings suggest that diversion of RE trafficking to the Golgi complex through a pathway involving SFKs, Cdc42, and Rab11a plays a general role in death signaling by mediating regulated changes in Golgi dynamics.


Assuntos
Endocitose , Endossomos/enzimologia , Complexo de Golgi/enzimologia , Membranas Intracelulares/enzimologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo , Actinas/metabolismo , Transporte Biológico/efeitos dos fármacos , Caspases/metabolismo , Compartimento Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Humanos , Membranas Intracelulares/efeitos dos fármacos , Modelos Biológicos , Transdução de Sinais/efeitos dos fármacos , Estaurosporina/farmacologia , Proteínas Virais/metabolismo
11.
J Biol Chem ; 283(49): 34352-64, 2008 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-18818208

RESUMO

The adenovirus type 2 Early Region 4 ORF4 (E4orf4) protein induces a caspase-independent death program in tumor cells involving changes in actin dynamics that are functionally linked to cell killing. Because an increase in myosin II-based contractility is needed for the death of E4orf4-expressing cells, we have proposed that alteration of cytoskeletal tension is part of the signals engaging the death pathway. Yet the mechanisms involved are poorly defined. Herein, we show that the Jun N-terminal kinase JNK is activated in part through a pathway involving Src, Rho, and ROCK (Rho kinase) and contributes to dysregulate adhesion dynamics and to kill cells in response to E4orf4. JNK supports the formation of atypically robust focal adhesions, which are bound to the assembly of the peculiar actomyosin network typifying E4orf4-induced cell death and which are required for driving nuclear condensation. Remarkably, the dramatic enlargement of focal adhesions, actin remodeling, and cell death all rely on paxillin phosphorylation at Ser-178, which is induced by E4orf4 in a JNK-dependent way. Furthermore, we found that Ser-178-paxillin phosphorylation is necessary to decrease adhesion turnover and to enhance the time residency of paxillin at focal adhesions, promoting its recruitment from an internal pool. Our results indicate that perturbation of tensional homeostasis by E4orf4 involves JNK-regulated changes in paxillin adhesion dynamics that are required to engage the death pathway. Moreover, our findings support a role for JNK-mediated paxillin phosphorylation in adhesion growth and stabilization during tension signaling.


Assuntos
MAP Quinase Quinase 4/metabolismo , Paxilina/química , Proteínas Virais/química , Actinas/metabolismo , Adenoviridae/metabolismo , Adesão Celular , Morte Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Vetores Genéticos , Homeostase , Humanos , Modelos Biológicos , Fosforilação , Quinases da Família src/metabolismo
12.
Mol Biol Cell ; 17(7): 3329-44, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16687574

RESUMO

The adenovirus early region 4 ORF4 protein (E4orf4) triggers a novel death program that bypasses classical apoptotic pathways in human cancer cells. Deregulation of the cell cytoskeleton is a hallmark of E4orf4 killing that relies on Src family kinases and E4orf4 phosphorylation. However, the cytoskeletal targets of E4orf4 and their role in the death process are unknown. Here, we show that E4orf4 translocates to cytoplasmic sites and triggers the assembly of a peculiar juxtanuclear actin-myosin network that drives polarized blebbing and nuclear shrinkage. We found that E4orf4 activates the myosin II motor and triggers de novo actin polymerization in the perinuclear region, promoting endosomes recruitment to the sites of actin assembly. E4orf4-induced actin dynamics requires interaction with Src family kinases and involves a spatial regulation of the Rho GTPases pathways Cdc42/N-Wasp, RhoA/Rho kinase, and Rac1, which make distinct contributions. Remarkably, activation of the Rho GTPases is required for induction of apoptotic-like cell death. Furthermore, inhibition of actin dynamics per se dramatically impairs E4orf4 killing. This work provides strong support for a causal role for endosome-associated actin dynamics in E4orf4 killing and in the regulation of cancer cell fate.


Assuntos
Actinas/metabolismo , Apoptose , Endossomos/metabolismo , Neoplasias/metabolismo , Proteínas Virais/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Actinas/antagonistas & inibidores , Animais , Apoptose/genética , Núcleo Celular/metabolismo , Ativação Enzimática , Humanos , Miosina Tipo II/metabolismo , Neoplasias/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Vesículas Transportadoras/enzimologia , Vesículas Transportadoras/fisiologia , Células Tumorais Cultivadas , Proteínas Virais/genética , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo
13.
J Biol Chem ; 279(24): 25905-15, 2004 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-15070897

RESUMO

Adenovirus type 2 (Ad2) early region 4 ORF4 (E4orf4) triggers a major death pathway that requires its accumulation in cellular membranes and its tyrosine phosphorylation. This program is regulated by Src family kinases and triggers a potent ZVAD (benzyloxycarbonyl-VAD)- and Bcl2-resistant cell death response in human-transformed cells. How E4orf4 deregulates Src-dependent signaling is unknown. Here we provide strong evidence that a physical interaction requiring the kinase domain of Src and the arginine-rich motif of E4orf4 is involved. The Src binding domain of E4orf4 overlaps with, but is distinct from that of the Balpha subunit of protein phosphatase 2A (PP2A-Balpha) and some E4orf4 complexes contain both PP2A and Src. Functional assays using mutant E4orf4 revealed that deregulation of Src signaling, activation of the Jun kinase pathway, and cell blebbing were all critically dependent on Src binding. In contrast, PP2A-Balpha binding per se was not required to engage the Src-dependent death pathway but was more critical for triggering a distinct death activity. Both E4orf4 death activities were manifested within a given cell population, were typified by distinct morphological features, and contributed to overall cell killing, although to different extents in various cell types. We conclude that E4orf4 binding to the Src kinase domain leads to deregulation of Src signaling and plays a crucial role in induction of the cytoplasmic death pathway. Nonetheless, both Src and PP2A enzymes are critical targets of E4orf4 that likely cooperate to trigger E4orf4-induced tumor cell killing and whose relative contributions may vary in function of the cellular background.


Assuntos
Proteínas E4 de Adenovirus/metabolismo , Proteínas Virais/metabolismo , Domínios de Homologia de src , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Morte Celular , Células Cultivadas , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Dados de Sequência Molecular , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Proteína Fosfatase 2 , Transdução de Sinais , Proteínas Virais/química
14.
J Cell Biol ; 158(3): 519-28, 2002 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-12163473

RESUMO

In transformed cells, induction of apoptosis by adenovirus type 2 (Ad2) early region 4 ORF 4 (E4orf4) correlates with accumulation of E4orf4 in the cell membrane-cytoskeleton fraction. However, E4orf4 is largely expressed in nuclear regions before the onset of apoptosis. To determine the relative contribution of nuclear E4orf4 versus membrane-associated E4orf4 to cell death signaling, we engineered green fluorescent fusion proteins to target E4orf4 to specific cell compartments. The targeting of Ad2 E4orf4 to cell membranes through a CAAX-box or a myristylation consensus signal sufficed to mimic the fast Src-dependent apoptotic program induced by wild-type E4orf4. In marked contrast, the nuclear targeting of E4orf4 abolished the early induction of extranuclear apoptosis. However, nuclear E4orf4 still induced a delayed cell death response independent of Src-like activity and of E4orf4 tyrosine phosphorylation. The zVAD.fmk-inhibitable caspases were dispensable for execution of both cell death programs. Nevertheless, both pathways led to caspase activation in some cell types through the mitochondrial pathway. Finally, our data support a critical role for calpains upstream in the death effector pathway triggered by the Src-mediated cytoplasmic death signal. We conclude that Ad2 E4orf4 induces two distinct cell death responses, whose relative contributions to cell killing may be determined by the genetic background.


Assuntos
Infecções por Adenoviridae/metabolismo , Adenoviridae/metabolismo , Apoptose/fisiologia , Células Eucarióticas/metabolismo , Transdução de Sinais/fisiologia , Proteínas Virais/metabolismo , Infecções por Adenoviridae/fisiopatologia , Apoptose/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/metabolismo , Calpaína/genética , Calpaína/metabolismo , Inibidores de Caspase , Caspases/genética , Caspases/metabolismo , Linhagem Celular Transformada , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Fragmentação do DNA/efeitos dos fármacos , Fragmentação do DNA/fisiologia , Inibidores Enzimáticos/farmacologia , Células Eucarióticas/citologia , Células Eucarióticas/efeitos dos fármacos , Proteínas de Fluorescência Verde , Humanos , Indicadores e Reagentes , Proteínas Luminescentes , Modelos Biológicos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Virais/genética , Proteína X Associada a bcl-2 , Quinases da Família src/efeitos dos fármacos , Quinases da Família src/metabolismo
15.
Mol Cell Biol ; 22(1): 41-56, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11739721

RESUMO

In transformed cells, the adenovirus E4orf4 death factor works in part by inducing a Src-mediated cytoplasmic apoptotic signal leading to caspase-independent membrane blebbing and cell death. Here we show that Src-family kinases modulate E4orf4 phosphorylation on tyrosine residues. Mutation of tyrosines 26, 42, and 59 to phenylalanines inhibited Src-induced phosphorylation of E4orf4 in vivo and in vitro but had no effect on the molecular association of E4orf4 with Src. However, in contrast to wild-type E4orf4, the nonphosphorylatable E4orf4 mutant was unable to modulate Src-dependent phosphorylation and was deficient in recruiting a subset of tyrosine-phosphorylated proteins. Indeed, the Src substrates cortactin and p62dok were found to associate with wild-type E4orf4 but not with the nonphosphorylatable E4orf4. Importantly, the nonphosphorylatable mutant E4orf4 was preferentially distributed in the cell nucleus, was unable to induce membrane blebbing, and had a highly impaired killing activity. Conversely, an activated form of E4orf4 was obtained by mutation of tyrosine 42 to glutamic acid. This pseudophosphorylated mutant E4orf4 was enriched in the cytoplasm and plasma membrane, showed increased binding to phosphotyrosine-containing proteins, and induced a dramatic blebbing phenotype associated with increased cell death. Altogether, our findings strongly suggest that Src-mediated phosphorylation of adenovirus type 2 E4orf4 is critical to promoting its cytoplasmic and membrane localization and is required for the transduction of E4orf4-Src-dependent induction of membrane blebbing. We propose that E4orf4 acts in part by uncoupling Src-dependent signals to drive the formation of a signaling complex that triggers a cytoplasmic death signal.


Assuntos
Adenoviridae/fisiologia , Apoptose/fisiologia , Transdução de Sinais/fisiologia , Proteínas Virais/metabolismo , Quinases da Família src/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/patologia , Tamanho Celular , Genes Reporter , Humanos , Immunoblotting , Microscopia Confocal , Modelos Biológicos , Fosforilação , Testes de Precipitina , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Tirosina/metabolismo , Proteínas Virais/genética
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