RESUMO
The kinases PERK and IRE1 alleviate endoplasmic reticulum (ER) stress by orchestrating the unfolded protein response (UPR). If stress mitigation fails, PERK promotes cell death by activating pro-apoptotic genes, including death receptor 5 (DR5). Conversely, IRE1-which harbors both kinase and endoribonuclease (RNase) modules-blocks apoptosis through regulated IRE1-dependent decay (RIDD) of DR5 mRNA. Under irresolvable ER stress, PERK activity persists, whereas IRE1 paradoxically attenuates, by mechanisms that remain obscure. Here, we report that PERK governs IRE1's attenuation through a phosphatase known as RPAP2 (RNA polymerase II-associated protein 2). RPAP2 reverses IRE1 phosphorylation, oligomerization, and RNase activation. This inhibits IRE1-mediated adaptive events, including activation of the cytoprotective transcription factor XBP1s, and ER-associated degradation of unfolded proteins. Furthermore, RIDD termination by RPAP2 unleashes DR5-mediated caspase activation and drives cell death. Thus, PERK attenuates IRE1 via RPAP2 to abort failed ER-stress adaptation and trigger apoptosis.
Assuntos
Apoptose/genética , Proteínas de Transporte/genética , Endorribonucleases/genética , Proteínas Serina-Treonina Quinases/genética , Resposta a Proteínas não Dobradas , eIF-2 Quinase/genética , Proteínas de Transporte/metabolismo , Caspases/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/genética , Endorribonucleases/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Transdução de Sinais , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo , eIF-2 Quinase/metabolismoRESUMO
Epithelial-to-mesenchymal transition (EMT) is a cellular process essential to the development and maintenance of solid tissues. In cancer, EMT suppresses apoptosis, but the mechanisms remain unclear. EMT selectively attenuated apoptosis signaling via the death receptors DR4 and DR5. Loss of the epithelial cell adhesion protein E-cadherin recapitulated this outcome, whereas homotypic E-cadherin engagement promoted apoptotic signaling via DR4/DR5, but not Fas. Depletion of α-catenin, which couples E-cadherin to the actin cytoskeleton, or actin polymerization inhibitors similarly attenuated DR4/DR5-induced apoptosis. E-cadherin bound specifically to ligated DR4/DR5, requiring extracellular cadherin domain 1 and calcium. E-cadherin augmented DR4/DR5 clustering and assembly of the death-inducing signaling complex (DISC), increasing caspase-8 activation in high molecular weight cell fractions. Conversely, EMT attenuated DR4/DR5-mediated DISC formation and caspase-8 stimulation. Consistent with these findings, epithelial cancer cell lines expressing higher E-cadherin levels displayed greater sensitivity to DR4/DR5-mediated apoptosis. These results have potential implications for tissue homeostasis as well as cancer therapy.
Assuntos
Apoptose/fisiologia , Caderinas/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Citoesqueleto de Actina/metabolismo , Antígenos CD , Proteínas Reguladoras de Apoptose/metabolismo , Caderinas/genética , Cálcio , Caspase 8/metabolismo , Linhagem Celular Tumoral , Citoesqueleto , Proteína de Domínio de Morte Associada a Fas/genética , Células HEK293 , Humanos , Interferência de RNA , RNA Interferente Pequeno , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Transdução de Sinais , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Fator de Crescimento Transformador beta/farmacologia , alfa Catenina/genéticaRESUMO
Multiple myeloma (MM) arises from malignant immunoglobulin (Ig)-secreting plasma cells and remains an incurable, often lethal disease despite therapeutic advances. The unfolded-protein response sensor IRE1α supports protein secretion by deploying a kinase-endoribonuclease module to activate the transcription factor XBP1s. MM cells may co-opt the IRE1α-XBP1s pathway; however, the validity of IRE1α as a potential MM therapeutic target is controversial. Genetic disruption of IRE1α or XBP1s, or pharmacologic IRE1α kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice and augmented efficacy of two established frontline antimyeloma agents, bortezomib and lenalidomide. Mechanistically, IRE1α perturbation inhibited expression of key components of the endoplasmic reticulum-associated degradation machinery, as well as secretion of Ig light chains and of cytokines and chemokines known to promote MM growth. Selective IRE1α kinase inhibition reduced viability of CD138+ plasma cells while sparing CD138- cells derived from bone marrows of newly diagnosed or posttreatment-relapsed MM patients, in both US- and European Union-based cohorts. Effective IRE1α inhibition preserved glucose-induced insulin secretion by pancreatic microislets and viability of primary hepatocytes in vitro, as well as normal tissue homeostasis in mice. These results establish a strong rationale for developing kinase-directed inhibitors of IRE1α for MM therapy.
Assuntos
Endorribonucleases/genética , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/genética , Idoso , Animais , Bortezomib/farmacologia , Estresse do Retículo Endoplasmático/genética , Endorribonucleases/antagonistas & inibidores , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lenalidomida/farmacologia , Masculino , Camundongos , Pessoa de Meia-Idade , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genética , Proteína 1 de Ligação a X-Box/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Apoptotic caspase activation mechanisms are well defined, yet inactivation modes remain unclear. The death receptors (DRs), DR4, DR5, and Fas, transduce cell-extrinsic apoptotic signals by recruiting caspase-8 into a death-inducing signaling complex (DISC). At the DISC, Cullin3-dependent polyubiquitination on the small catalytic subunit of caspase-8 augments stimulation. Here we report that tumor necrosis factor receptor-associated factor 2 (TRAF2) interacts with caspase-8 at the DISC, downstream of Cullin3. TRAF2 directly mediates RING-dependent, K48-linked polyubiquitination on the large catalytic domain of caspase-8. This modification destines activated caspase-8 molecules to rapid proteasomal degradation upon autoprocessing and cytoplasmic translocation. TRAF2 depletion lowers the signal threshold for DR-mediated apoptosis, altering cell life versus death decisions in vitro and in vivo. Thus, TRAF2 sets a critical barrier for cell-extrinsic apoptosis commitment by tagging activated caspase-8 with a K48-ubiquitin shutoff timer. These results may have important implications for caspase regulation mechanisms.
Assuntos
Apoptose , Caspase 8/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Fator 2 Associado a Receptor de TNF/fisiologia , Sequência de Aminoácidos , Animais , Domínio Catalítico , Sobrevivência Celular , Proteínas Culina/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Ativação Enzimática , Células HCT116 , Humanos , Leupeptinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Mapeamento de Peptídeos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , UbiquitinaçãoRESUMO
TNF superfamily death ligands are expressed on the surface of immune cells and can trigger apoptosis in susceptible cancer cells by engaging cognate death receptors. A recombinant soluble protein comprising the ectodomain of Apo2 ligand/TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) has shown remarkable preclinical anticancer activity but lacked broad efficacy in patients, possibly owing to insufficient exposure or potency. We observed that antibody cross-linking substantially enhanced cytotoxicity of soluble Apo2L/TRAIL against diverse cancer cell lines. Presentation of the ligand on glass-supported lipid bilayers enhanced its ability to drive receptor microclustering and apoptotic signaling. Furthermore, covalent surface attachment of Apo2L/TRAIL onto liposomes--synthetic lipid-bilayer nanospheres--similarly augmented activity. In vivo, liposome-displayed Apo2L/TRAIL achieved markedly better exposure and antitumor activity. Thus, covalent synthetic-membrane attachment of a cell-surface ligand enhances efficacy, increasing therapeutic potential. These findings have translational implications for liposomal approaches as well as for Apo2L/TRAIL and other clinically relevant TNF ligands.
Assuntos
Antineoplásicos/química , Membrana Celular/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Apoptose , Biotinilação , Ligante CD27/metabolismo , Caspase 8/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral , Epitopos/química , Proteína Ligante Fas/metabolismo , Humanos , Sistema Imunitário , Imunoterapia/métodos , Concentração Inibidora 50 , Ligantes , Lipossomos/química , Camundongos , Camundongos Nus , Microscopia de Fluorescência , Transplante de Neoplasias , Neoplasias/imunologia , Neoplasias/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Adjuvants are an essential component of modern vaccines and used for their ability to elicit immunity to coadministered Ags. Many adjuvants in clinical development are particulates, but how they drive innate and adaptive immune responses remains poorly understood. Studies have shown that a number of vaccine adjuvants activate inflammasome pathways in isolated APCs. However, the contribution of inflammasome activation to vaccine-mediated immunity in vivo remains controversial. In this study, we evaluated immune cell responses to the ISCOMATRIX adjuvant (IMX) in mice. Like other particulate vaccine adjuvants, IMX potently activated the NALP-3-ASC-Caspase-1 inflammasome in APCs, leading to IL-1ß and IL-18 production. The IL-18R pathway, but not IL-1R, was required for early innate and subsequent cellular immune responses to a model IMX vaccine. APCs directly exposed to IMX underwent an endosome-mediated cell-death response, which we propose initiates inflammatory events locally at the injection site. Importantly, both inflammasome-related and -unrelated pathways contributed to IL-18 dependence in vivo following IMX administration. TNF-α provided a physiological priming signal for inflammasome-dependent IL-18 production by APCs, which correlated with reduced vaccine-mediated immune cell responses in TNF-α- or TNFR-deficient mice. Taken together, our findings highlight an important disconnect between the mechanisms of vaccine adjuvant action in vitro versus in vivo.
Assuntos
Colesterol/imunologia , Imunidade/imunologia , Inflamassomos/imunologia , Interleucina-18/imunologia , Fosfolipídeos/imunologia , Saponinas/imunologia , Trifosfato de Adenosina/imunologia , Trifosfato de Adenosina/metabolismo , Adjuvantes Imunológicos/farmacologia , Animais , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Colesterol/farmacologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Combinação de Medicamentos , Humanos , Imunidade/efeitos dos fármacos , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Lisossomos/efeitos dos fármacos , Lisossomos/imunologia , Lisossomos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Fosfolipídeos/farmacologia , Receptores do Fator de Necrose Tumoral/deficiência , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/imunologia , Saponinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/deficiência , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
During the course of biopharmaceutical production, heterologous protein expression in Chinese hamster ovary (CHO) cells imposes a high proteostatic burden that requires cellular adaptation. To mitigate such burden, cells utilize the unfolded protein response (UPR), which increases endoplasmic reticulum (ER) capacity to accommodate elevated rates of protein synthesis and folding. In this study, we show that during production the UPR regulates growth factor signaling to modulate growth and protein synthesis. Specifically, the protein kinase R-like ER kinase (PERK) branch of the UPR is responsible for transcriptional down-regulation of platelet-derived growth factor receptor alpha (PDGFRa) and attenuation of the IRE1-alpha (IRE1a) branch of the UPR. PERK knockout (KO) cell lines displayed reduced growth and viability due to higher rates of apoptosis despite having stabilized PDGFRa levels. Knocking out PERK in an apoptosis impaired (Bax/Bak double KO) antibody-expressing cell line prevented apoptotic cell death and revealed that apoptosis was likely triggered by increased ER stress and reactive oxygen species levels in the PERK KO hosts. Our findings suggest that attenuation of IRE1a and PDGFRa signaling by the PERK branch of the UPR reduces ER protein folding capacity and hence specific productivity of CHO cells in order to mitigate UPR and prevent apoptotic cell death. Last, Bax/Bak/PERK triple KO CHO cell lines displayed 2-3 folds higher specific productivity and titer (up to 8 g/L), suggesting that modulation of PERK signaling during production processes can greatly improve specific productivity in CHO cells.
RESUMO
Although the signal transduction mechanisms of the receptor tyrosine kinase MET are well defined, less is known about its close relative RON. MET initiates intracellular signaling by autophosphorylation on specific cytoplasmic tyrosines that form docking sites for the adaptor proteins Grb2 and Gab1. Grb2 binds directly and is essential for all of the biological activities of MET. Gab1 docks either directly or indirectly via Grb2 and controls only a subset of MET functions. Because MET and RON possess similar adaptor binding sites, it was anticipated that their adaptor interactions would be conserved. Here we show that in contrast to MET, RON relies primarily on Gab1 for signal transmission. Surprisingly, disruption of the Grb2 docking site of RON or Grb2 depletion augments activity, whereas enhancement of Grb2 binding attenuates Gab1 recruitment and signaling. Hence, RON and MET differ in their adaptor interactions; furthermore, Grb2 performs a novel antagonistic role in the context of RON signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína Adaptadora GRB2/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Proteína Adaptadora GRB2/genética , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Fosfoproteínas/genética , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-met/genética , Receptores Proteína Tirosina Quinases/genéticaRESUMO
The unfolded protein response (UPR) maintains homeostasis of the endoplasmic reticulum (ER). Residing in the ER membrane, the UPR mediator Ire1 deploys its cytoplasmic kinase-endoribonuclease domain to activate the key UPR transcription factor Xbp1 through non-conventional splicing of Xbp1 mRNA. Ire1 also degrades diverse ER-targeted mRNAs through regulated Ire1-dependent decay (RIDD), but how it spares Xbp1 mRNA from this decay is unknown. Here, we identify binding sites for the RNA-binding protein Pumilio in the 3'UTR Drosophila Xbp1. In the developing Drosophila eye, Pumilio binds both the Xbp1unspliced and Xbp1spliced mRNAs, but only Xbp1spliced is stabilized by Pumilio. Furthermore, Pumilio displays Ire1 kinase-dependent phosphorylation during ER stress, which is required for its stabilization of Xbp1spliced. hIRE1 can phosphorylate Pumilio directly, and phosphorylated Pumilio protects Xbp1spliced mRNA against RIDD. Thus, Ire1-mediated phosphorylation enables Pumilio to shield Xbp1spliced from RIDD. These results uncover an unexpected regulatory link between an RNA-binding protein and the UPR.
Assuntos
Proteínas de Drosophila , Proteínas Serina-Treonina Quinases , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Estresse do Retículo Endoplasmático/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/metabolismo , Resposta a Proteínas não Dobradas/genética , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
Dendritic cells (DCs) promote adaptive immunity by cross-presenting antigen-based epitopes to CD8+ T cells. DCs process internalized protein antigens into peptides that enter the endoplasmic reticulum (ER), bind to major histocompatibility type I (MHC-I) protein complexes, and are transported to the cell surface for cross-presentation. DCs can exhibit activation of the ER stress sensor IRE1α without ER stress, but the underlying mechanism remains obscure. Here, we show that antigen-derived hydrophobic peptides can directly engage ER-resident IRE1α, masquerading as unfolded proteins. IRE1α activation depletes MHC-I heavy-chain mRNAs through regulated IRE1α-dependent decay (RIDD), curtailing antigen cross-presentation. In tumor-bearing mice, IRE1α disruption increased MHC-I expression on tumor-infiltrating DCs and enhanced recruitment and activation of CD8+ T cells. Moreover, IRE1α inhibition synergized with anti-PD-L1 antibody treatment to cause tumor regression. Our findings identify an unexpected cell-biological mechanism of antigen-driven IRE1α activation in DCs, revealing translational potential for cancer immunotherapy.
Assuntos
Apresentação Cruzada , Células Dendríticas , Estresse do Retículo Endoplasmático , Endorribonucleases , Neoplasias , Proteínas Serina-Treonina Quinases , Animais , Apresentação de Antígeno , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Endorribonucleases/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Camundongos , Neoplasias/imunologia , Neoplasias/metabolismo , Peptídeos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Asthma and inflammatory airway diseases restrict airflow in the lung, compromising gas exchange and lung function. Inhaled corticosteroids (ICSs) can reduce inflammation, control symptoms, and improve lung function; however, a growing number of patients with severe asthma do not benefit from ICS. Using bronchial airway epithelial brushings from patients with severe asthma or primary human cells, we delineated a corticosteroid-driven fibroblast growth factor (FGF)-dependent inflammatory axis, with FGF-responsive fibroblasts promoting downstream granulocyte colony-stimulating factor (G-CSF) production, hyaluronan secretion, and neutrophilic inflammation. Allergen challenge studies in mice demonstrate that the ICS, fluticasone propionate, inhibited type 2-driven eosinophilia but induced a concomitant increase in FGFs, G-CSF, hyaluronan, and neutrophil infiltration. We developed a model of steroid-induced neutrophilic inflammation mediated, in part, by induction of an FGF-dependent epithelial-mesenchymal axis, which may explain why some individuals do not benefit from ICS. In further proof-of-concept experiments, we found that combination therapy with pan-FGF receptor inhibitors and corticosteroids prevented both eosinophilic and steroid-induced neutrophilic inflammation. Together, these results establish FGFs as therapeutic targets for severe asthma patients who do not benefit from ICS.
Assuntos
Asma , Fatores de Crescimento de Fibroblastos , Corticosteroides/farmacologia , Corticosteroides/uso terapêutico , Animais , Fluticasona/farmacologia , Fluticasona/uso terapêutico , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Humanos , Ácido Hialurônico , Inflamação/tratamento farmacológico , CamundongosRESUMO
Fibrosis is the major risk factor associated with morbidity and mortality in patients with non-alcoholic steatohepatitis (NASH)-driven chronic liver disease. Although numerous efforts have been made to identify the mediators of the initiation of liver fibrosis, the molecular underpinnings of fibrosis progression remain poorly understood, and therapies to arrest liver fibrosis progression are elusive. Here, we identify a pathway involving WNT1-inducible signaling pathway protein 1 (WISP1) and myocardin-related transcription factor (MRTF) as a central mechanism driving liver fibrosis progression through the integrin-dependent transcriptional reprogramming of myofibroblast cytoskeleton and motility. In mice, WISP1 deficiency protects against fibrosis progression, but not fibrosis onset. Moreover, the therapeutic administration of a novel antibody blocking WISP1 halted the progression of existing liver fibrosis in NASH models. These findings implicate the WISP1-MRTF axis as a crucial determinant of liver fibrosis progression and support targeting this pathway by antibody-based therapy for the treatment of NASH fibrosis.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Fatores de Transcrição , Animais , Fígado/metabolismo , Cirrose Hepática/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismo , Proteínas Nucleares , Transdução de Sinais , Transativadores , Fatores de Transcrição/metabolismoRESUMO
Inositol requiring enzyme 1 (IRE1) mitigates endoplasmic-reticulum (ER) stress by orchestrating the unfolded-protein response (UPR). IRE1 spans the ER membrane, and signals through a cytosolic kinase-endoribonuclease module. The endoribonuclease generates the transcription factor XBP1s by intron excision between similar RNA stem-loop endomotifs, and depletes select cellular mRNAs through regulated IRE1-dependent decay (RIDD). Paradoxically, in mammals RIDD seems to target only mRNAs with XBP1-like endomotifs, while in flies RIDD exhibits little sequence restriction. By comparing nascent and total IRE1α-controlled mRNAs in human cells, we identify not only canonical endomotif-containing RIDD substrates, but also targets without such motifs-degraded by a process we coin RIDDLE, for RIDD lacking endomotif. IRE1α displays two basic endoribonuclease modalities: highly specific, endomotif-directed cleavage, minimally requiring dimers; and more promiscuous, endomotif-independent processing, requiring phospho-oligomers. An oligomer-deficient IRE1α mutant fails to support RIDDLE in vitro and in cells. Our results advance current mechanistic understanding of the UPR.
Assuntos
Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Retículo Endoplasmático/genética , Endorribonucleases/genética , Humanos , Proteínas Serina-Treonina Quinases/genética , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Resposta a Proteínas não DobradasRESUMO
Disruption of protein folding in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR)-a signaling network that ultimately determines cell fate. Initially, UPR signaling aims at cytoprotection and restoration of ER homeostasis; that failing, it drives apoptotic cell death. ER stress initiates apoptosis through intracellular activation of death receptor 5 (DR5) independent of its canonical extracellular ligand Apo2L/TRAIL; however, the mechanism underlying DR5 activation is unknown. In cultured human cells, we find that misfolded proteins can directly engage with DR5 in the ER-Golgi intermediate compartment, where DR5 assembles pro-apoptotic caspase 8-activating complexes. Moreover, peptides used as a proxy for exposed misfolded protein chains selectively bind to the purified DR5 ectodomain and induce its oligomerization. These findings indicate that misfolded proteins can act as ligands to activate DR5 intracellularly and promote apoptosis. We propose that cells can use DR5 as a late protein-folding checkpoint before committing to a terminal apoptotic fate.
Proteins are chains of building blocks called amino acids, folded into a flexible 3D shape that is critical for its biological activity. This shape depends on many factors, but one is the chemistry of the amino acids. Because the internal and external environments of cells are mostly water-filled, correctly folded proteins often display so-called hydrophilic (or 'water-loving') amino acids on their surface, while tucking hydrophobic (or 'water-hating') amino acids on the inside. A compartment within the cell called the endoplasmic reticulum folds the proteins that are destined for the outside of the cell. It can handle a steady stream of protein chains, but a sudden increase in demand for production, or issues with the underlying machinery, can stress the endoplasmic reticulum and hinder protein folding. This is problematic because incorrectly folded proteins cannot work as they should and can be toxic to the cell that made them or even to other cells. Many cells handle this kind of stress by activating a failsafe alarm system called the unfolded protein response. It detects the presence of incorrectly shaped proteins and sends signals that try to protect the cell and restore protein folding to normal. If that fails within a certain period of time, it switches to signals that tell the cell to safely self-destruct. That switch, from protection to self-destruction, involves a protein called death receptor 5, or DR5 for short. DR5 typically triggers the cell's self-destruct program by forming molecular clusters at the cell's surface, in response to a signal it receives from the exterior. During a failed unfolded protein response, DR5 seems instead to act in response to signals from inside the cell, but it was not clear how this works. To find out, Lam et al. stressed the endoplasmic reticulum in human cells by forcing it to fold a lot of proteins. This revealed that DR5 sticks to misfolded proteins when they leave the endoplasmic reticulum. In response, DR5 molecules form clusters that trigger the cell's self-destruct program. DR5 directly recognized hydrophobic amino acids on the misfolded protein's surface that would normally be hidden inside. When Lam et al. edited these hydrophobic regions to become hydrophilic, the DR5 molecules could no longer detect them as well. This stopped the cells from dying so easily when they were under stress. It seems that DR5 decides the fate of the cell by detecting proteins that were incorrectly folded in the endoplasmic reticulum. Problems with protein folding occur in many human diseases, including metabolic conditions, cancer and degenerative brain disorders. Future work could reveal whether controlling the activation of DR5 could help to influence if and when cells die. The next step is to understand how DR5 interacts with incorrectly folded proteins at the atomic level. This could aid the design of drugs that specifically target such receptors.
Assuntos
Apoptose/genética , Estresse do Retículo Endoplasmático , Dobramento de Proteína , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Resposta a Proteínas não Dobradas , Células HCT116 , Células Hep G2 , Humanos , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
Cancer cells exploit the unfolded protein response (UPR) to mitigate endoplasmic reticulum (ER) stress caused by cellular oncogene activation and a hostile tumor microenvironment (TME). The key UPR sensor IRE1α resides in the ER and deploys a cytoplasmic kinase-endoribonuclease module to activate the transcription factor XBP1s, which facilitates ER-mediated protein folding. Studies of triple-negative breast cancer (TNBC)-a highly aggressive malignancy with a dismal posttreatment prognosis-implicate XBP1s in promoting tumor vascularization and progression. However, it remains unknown whether IRE1α adapts the ER in TNBC cells and modulates their TME, and whether IRE1α inhibition can enhance antiangiogenic therapy-previously found to be ineffective in patients with TNBC. To gauge IRE1α function, we defined an XBP1s-dependent gene signature, which revealed significant IRE1α pathway activation in multiple solid cancers, including TNBC. IRE1α knockout in TNBC cells markedly reversed substantial ultrastructural expansion of their ER upon growth in vivo. IRE1α disruption also led to significant remodeling of the cellular TME, increasing pericyte numbers while decreasing cancer-associated fibroblasts and myeloid-derived suppressor cells. Pharmacologic IRE1α kinase inhibition strongly attenuated growth of cell line-based and patient-derived TNBC xenografts in mice and synergized with anti-VEGFA treatment to cause tumor stasis or regression. Thus, TNBC cells critically rely on IRE1α to adapt their ER to in vivo stress and to adjust the TME to facilitate malignant growth. TNBC reliance on IRE1α is an important vulnerability that can be uniquely exploited in combination with antiangiogenic therapy as a promising new biologic approach to combat this lethal disease. SIGNIFICANCE: Pharmacologic IRE1α kinase inhibition reverses ultrastructural distension of the ER, normalizes the tumor vasculature, and remodels the cellular TME, attenuating TNBC growth in mice.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos Imunológicos/farmacologia , Estresse do Retículo Endoplasmático/fisiologia , Endorribonucleases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/terapia , Animais , Antineoplásicos Imunológicos/imunologia , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endorribonucleases/genética , Feminino , Técnicas de Inativação de Genes , Humanos , Camundongos , Camundongos SCID , Neovascularização Patológica/terapia , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , Neoplasias de Mama Triplo Negativas/irrigação sanguínea , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/imunologia , Proteína 1 de Ligação a X-Box/antagonistas & inibidores , Proteína 1 de Ligação a X-Box/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Upon detecting endoplasmic reticulum (ER) stress, the unfolded protein response (UPR) orchestrates adaptive cellular changes to reestablish homeostasis. If stress resolution fails, the UPR commits the cell to apoptotic death. Here we show that in hematopoietic cells, including multiple myeloma (MM), lymphoma, and leukemia cell lines, ER stress leads to caspase-mediated cleavage of the key UPR sensor IRE1 within its cytoplasmic linker region, generating a stable IRE1 fragment comprising the ER-lumenal domain and transmembrane segment (LDTM). This cleavage uncouples the stress-sensing and signaling domains of IRE1, attenuating its activation upon ER perturbation. Surprisingly, LDTM exerts negative feedback over apoptotic signaling by inhibiting recruitment of the key proapoptotic protein BAX to mitochondria. Furthermore, ectopic LDTM expression enhances xenograft growth of MM tumors in mice. These results uncover an unexpected mechanism of cross-regulation between the apoptotic caspase machinery and the UPR, which has biologically significant consequences for cell survival under ER stress.
Assuntos
Apoptose , Caspases/metabolismo , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Linhagem Celular , Humanos , Camundongos , ProteóliseRESUMO
Agonism of members of the tumor necrosis factor receptor superfamily (TNFRSF) with monoclonal antibodies is of high therapeutic interest due to their role in immune regulation and cell proliferation. A major hurdle for pharmacologic activation of this receptor class is the requirement for high-order clustering, a mechanism that imposes a reliance in vivo on Fc receptor-mediated crosslinking. This extrinsic dependence represents a potential limitation of virtually the entire pipeline of agonist TNFRSF antibody drugs, of which none have thus far been approved or reached late-stage clinical trials. We show that tetravalent biepitopic targeting enables robust intrinsic antibody agonism for two members of this family, OX40 and DR5, that is superior to extrinsically crosslinked native parental antibodies. Tetravalent biepitopic anti-OX40 engagement co-stimulated OX40low cells, obviated the requirement for CD28 co-signal for T cell activation, and enabled superior pharmacodynamic activity relative to native IgG in a murine vaccination model. This work establishes a proof of concept for an engineering approach that addresses a major gap for the therapeutic activation of this important receptor class.
Assuntos
Anticorpos Monoclonais/imunologia , Capeamento Imunológico , Ligante OX40/agonistas , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Animais , Antígenos CD28/imunologia , Células CHO , Cricetulus , Humanos , Células Jurkat , Camundongos , Camundongos SCID , Camundongos Transgênicos , Ligante OX40/imunologia , Receptores Fc/imunologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Linfócitos T/citologiaRESUMO
The cell-extrinsic apoptotic pathway triggers programmed cell death in response to certain ligands that bind to cell-surface death receptors. Apoptosis is essential for normal development and homeostasis in metazoans, and furthermore, selective activation of the cell-extrinsic pathway in tumor cells holds considerable promise for cancer therapy. We used phage display to identify peptides and synthetic antibodies that specifically bind to the human proapoptotic death receptor DR5. Despite great differences in overall size and structure, the DR5-binding peptides and antibodies shared a tripeptide motif, which was conserved within a disulfide-constrained loop of the peptides and the third complementarity determining region of the antibody heavy chains. The X-ray crystal structure of an antibody in complex with DR5 revealed that the tripeptide motif is buried at the core of the interface, confirming its central role in antigen recognition. We found that certain peptides and antibodies exhibited potent proapoptotic activity against DR5-expressing SK-MES-1 lung carcinoma cells. These phage-derived ligands may be useful for elucidating DR5 activation at the molecular level and for creating synthetic agonists of proapoptotic death receptors.
Assuntos
Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Fragmentos Fab das Imunoglobulinas/química , Oligopeptídeos/química , Receptores do Fator de Necrose Tumoral/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Fragmentos Fab das Imunoglobulinas/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/farmacologia , Biblioteca de Peptídeos , Conformação Proteica , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/agonistas , Receptores do Fator de Necrose Tumoral/imunologiaRESUMO
Apo2L/TRAIL is a member of the tumor necrosis factor superfamily and an important inducer of apoptosis. Recombinant human (rhu) Apo2L/TRAIL has been attractive as a potential cancer therapeutic because many types of tumor cells are sensitive to its apoptosis-inducing effects. Nonclinical toxicology studies were conducted to evaluate the safety of rhuApo2L/TRAIL for possible use in humans. The cynomolgus monkey was chosen for this safety assessment based on high protein sequence homology between human and cynomolgus Apo2L/TRAIL and comparable expression of their receptors. Although hepatotoxicity was observed in repeat-dose monkey studies with rhuApo2L/TRAIL, all animals that displayed hepatotoxicity had developed antitherapeutic antibodies (ATAs). The cynomolgus ATAs augmented the cytotoxicity of rhuApo2L/TRAIL but not of its cynomolgus counterpart. Of note, human and cynomolgus Apo2L/TRAIL differ by four amino acids, three of which are surface-exposed. In vivo studies comparing human and cynomolgus Apo2L/TRAIL supported the conclusion that these distinct amino acids served as epitopes for cross-species ATAs, capable of crosslinking rhuApo2L/TRAIL and thus triggering hepatocyte apoptosis. We describe a hapten-independent mechanism of immune-mediated, drug-related hepatotoxicity - in this case - associated with the administration of a human recombinant protein in monkeys. The elucidation of this mechanism enabled successful transition of rhuApo2L/TRAIL into human clinical trials.
Assuntos
Anticorpos/toxicidade , Anticorpos/uso terapêutico , Proteínas Recombinantes/toxicidade , Proteínas Recombinantes/uso terapêutico , Ligante Indutor de Apoptose Relacionado a TNF/toxicidade , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico , Animais , Modelos Animais de Doenças , Humanos , Células Jurkat , Rim/efeitos dos fármacos , Rim/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Macaca fascicularis , Especificidade da EspécieRESUMO
FGF receptors (FGFR) are attractive candidate targets for cancer therapy because they are dysregulated in several human malignancies. FGFR2 and FGFR3 can be inhibited potentially without disrupting adult tissue homeostasis. In contrast, blocking the closely related FGFR1 and FGFR4, which regulate specific metabolic functions, carries a greater safety risk. An anti-FGFR3 antibody was redesigned here to create function-blocking antibodies that bind with dual specificity to FGFR3 and FGFR2 but spare FGFR1 and FGFR4. R3Mab, a previously developed monospecific anti-FGFR3 antibody, was modified via structure-guided phage display and acquired additional binding to FGFR2. The initial variant was trispecific, binding tightly to FGFR3 and FGFR2 and moderately to FGFR4, while sparing FGFR1. The X-ray crystallographic structure indicated that the antibody variant was bound to a similar epitope on FGFR2 as R3Mab on FGFR3. The antibody was further engineered to decrease FGFR4-binding affinity while retaining affinity for FGFR3 and FGFR2. The resulting dual-specific antibodies blocked FGF binding to FGFR3 and FGFR2 and inhibited downstream signaling. Moreover, they displayed efficacy in mice against human tumor xenografts overexpressing FGFR3 or FGFR2. Thus, a monospecific antibody can be exquisitely tailored to confer or remove binding to closely related targets to expand and refine therapeutic potential.