RESUMEN
In metastatic urothelial cancer (mUC), cisplatin versus carboplatin leads to durable disease control in a subset of patients. The IMvigor130 trial reveals more favorable effects with atezolizumab combined with gemcitabine and cisplatin (GemCis) versus gemcitabine and carboplatin (GemCarbo). This study investigates the immunomodulatory effects of cisplatin as a potential explanation for these observations. Our findings indicate that improved outcomes with GemCis versus GemCarbo are primarily observed in patients with pretreatment tumors exhibiting features of restrained adaptive immunity. In addition, GemCis versus GemCarbo ± atezolizumab induces transcriptional changes in circulating immune cells, including upregulation of antigen presentation and T cell activation programs. In vitro experiments demonstrate that cisplatin, compared with carboplatin, exerts direct immunomodulatory effects on cancer cells, promoting dendritic cell activation and antigen-specific T cell killing. These results underscore the key role of immune modulation in cisplatin's efficacy in mUC and highlight the importance of specific chemotherapy backbones in immunotherapy combination regimens.
Asunto(s)
Anticuerpos Monoclonales Humanizados , Carcinoma de Células Transicionales , Neoplasias de la Vejiga Urinaria , Neoplasias Urológicas , Humanos , Carboplatino/uso terapéutico , Carcinoma de Células Transicionales/tratamiento farmacológico , Carcinoma de Células Transicionales/inducido químicamente , Carcinoma de Células Transicionales/patología , Cisplatino/uso terapéutico , Desoxicitidina/uso terapéutico , Gemcitabina , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/patología , Neoplasias Urológicas/tratamiento farmacológico , Neoplasias Urológicas/inducido químicamente , Neoplasias Urológicas/patologíaRESUMEN
Receptor interacting protein 1 (RIP1) kinase is a critical regulator of inflammation and cell death signaling, and plays a crucial role in maintaining immune responses and proper tissue homeostasis. Mounting evidence argues for the importance of RIP1 post-translational modifications in control of its function. Ubiquitination by E3 ligases, such as inhibitors of apoptosis (IAP) proteins and LUBAC, as well as the reversal of these modifications by deubiquitinating enzymes, such as A20 and CYLD, can greatly influence RIP1 mediated signaling. In addition, cleavage by caspase-8, RIP1 autophosphorylation, and phosphorylation by a number of signaling kinases can greatly impact cellular fate. Disruption of the tightly regulated RIP1 modifications can lead to signaling disbalance in TNF and/or TLR controlled and other inflammatory pathways, and result in severe human pathologies. This review will focus on RIP1 and its many modifications with an emphasis on ubiquitination, phosphorylation, and cleavage, and their functional impact on the RIP1's role in signaling pathways.
Asunto(s)
Procesamiento Proteico-Postraduccional , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Apoptosis/fisiología , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Fosforilación , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Transducción de Señal , UbiquitinaciónRESUMEN
Proper maintenance of organismal homeostasis, development, and immune defense requires precise regulation of survival and signaling pathways. Inhibitor of apoptosis (IAP) proteins are evolutionarily conserved regulators of cell death and immune signaling that impact numerous cellular processes. Although initially characterized as inhibitors of apoptosis, the ubiquitin ligase activity of IAP proteins is critical for modulating various signaling pathways (e.g., NF-κB, MAPK) and cell survival. Cellular IAP1 and 2 regulate the pro-survival canonical NF-κB pathway by ubiquitinating RIP1 and themselves thus enabling recruitment of kinase (IKK) and E3 ligase (LUBAC) complexes. On the other hand, c-IAP1 and c-IAP2 are negative regulators of noncanonical NF-κB signaling by promoting ubiquitination and consequent proteasomal degradation of the NF-κB-inducing kinase NIK. Here we describe the involvement of c-IAP1 and c-IAP2 in NF-κB signaling and provide detailed methodology for examining functional roles of c-IAPs in these pathways.
Asunto(s)
Transducción de Señal , Apoptosis , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , FN-kappa B/metabolismo , Receptores del Factor de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/metabolismo , UbiquitinaciónRESUMEN
RIP1 kinase-mediated inflammatory and cell death pathways have been implicated in the pathology of acute and chronic disorders of the nervous system. Here, we describe a novel animal model of RIP1 kinase deficiency, generated by knock-in of the kinase-inactivating RIP1(D138N) mutation in rats. Homozygous RIP1 kinase-dead (KD) rats had normal development, reproduction and did not show any gross phenotypes at baseline. However, cells derived from RIP1 KD rats displayed resistance to necroptotic cell death. In addition, RIP1 KD rats were resistant to TNF-induced systemic shock. We studied the utility of RIP1 KD rats for neurological disorders by testing the efficacy of the genetic inactivation in the transient middle cerebral artery occlusion/reperfusion model of brain injury. RIP1 KD rats were protected in this model in a battery of behavioral, imaging, and histopathological endpoints. In addition, RIP1 KD rats had reduced inflammation and accumulation of neuronal injury biomarkers. Unbiased proteomics in the plasma identified additional changes that were ameliorated by RIP1 genetic inactivation. Together these data highlight the utility of the RIP1 KD rats for target validation and biomarker studies for neurological disorders.
Asunto(s)
Lesiones Encefálicas/genética , Muerte Celular/genética , Isquemia/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Modelos Animales de Enfermedad , Masculino , Ratas , Ratas Sprague-Dawley , Proteína Serina-Treonina Quinasas de Interacción con ReceptoresRESUMEN
RIP1 kinase is proposed to play a critical role in driving necroptosis and inflammation in neurodegenerative disorders, including Amyotrophic Lateral Sclerosis (ALS). Preclinical studies indicated that while pharmacological inhibition of RIP1 kinase can ameliorate axonal pathology and delay disease onset in the mutant SOD1 transgenic (SOD1-Tg) mice, genetic blockade of necroptosis does not provide benefit in this mouse model. To clarify the role of RIP1 kinase activity in driving pathology in SOD1-Tg mice, we crossed SOD1-Tgs to RIP1 kinase-dead knock-in mice, and measured disease progression using functional and histopathological endpoints. Genetic inactivation of the RIP1 kinase activity in the SOD1-Tgs did not benefit the declining muscle strength or nerve function, motor neuron degeneration or neuroinflammation. In addition, we did not find evidence of phosphorylated RIP1 accumulation in the spinal cords of ALS patients. On the other hand, genetic inactivation of RIP1 kinase activity ameliorated the depletion of the neurotransmitter dopamine in a toxin model of dopaminergic neurodegeneration. These findings indicate that RIP1 kinase activity is dispensable for disease pathogenesis in the SOD1-Tg mice while inhibition of kinase activity may provide benefit in acute injury models.
Asunto(s)
Esclerosis Amiotrófica Lateral/enzimología , Proteínas Activadoras de GTPasa/genética , Neuronas Motoras/patología , Superóxido Dismutasa-1/genética , Esclerosis Amiotrófica Lateral/etiología , Esclerosis Amiotrófica Lateral/patología , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Células HT29 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , NecroptosisRESUMEN
The dysregulation of multiple signaling pathways, including those through endosomal Toll-like receptors (TLRs), Fc gamma receptors (FcγR), and antigen receptors in B cells (BCR), promote an autoinflammatory loop in systemic lupus erythematosus (SLE). Here, we used selective small-molecule inhibitors to assess the regulatory roles of interleukin-1 receptor (IL-1R)-associated kinase 4 (IRAK4) and Bruton's tyrosine kinase (BTK) in these pathways. The inhibition of IRAK4 repressed SLE immune complex- and TLR7-mediated activation of human plasmacytoid dendritic cells (pDCs). Correspondingly, the expression of interferon (IFN)-responsive genes (IRGs) in cells and in mice was positively regulated by the kinase activity of IRAK4. Both IRAK4 and BTK inhibition reduced the TLR7-mediated differentiation of human memory B cells into plasmablasts. TLR7-dependent inflammatory responses were differentially regulated by IRAK4 and BTK by cell type: In pDCs, IRAK4 positively regulated NF-κB and MAPK signaling, whereas in B cells, NF-κB and MAPK pathways were regulated by both BTK and IRAK4. In the pristane-induced lupus mouse model, inhibition of IRAK4 reduced the expression of IRGs during disease onset. Mice engineered to express kinase-deficient IRAK4 were protected from both chemical (pristane-induced) and genetic (NZB/W_F1 hybrid) models of lupus development. Our findings suggest that kinase inhibitors of IRAK4 might be a therapeutic in patients with SLE.
Asunto(s)
Células Dendríticas/metabolismo , Endosomas/metabolismo , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Glicoproteínas de Membrana/metabolismo , Células Plasmáticas/metabolismo , Transducción de Señal , Receptor Toll-Like 7/metabolismo , Agammaglobulinemia Tirosina Quinasa , Animales , Endosomas/genética , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/genética , Glicoproteínas de Membrana/genética , Ratones , Receptor Toll-Like 7/genéticaRESUMEN
Receptor interacting protein kinase 1 (RIP1) is a critical effector of inflammatory responses and cell death activation. Cell death pathways regulated by RIP1 include caspase-dependent apoptosis and caspase-independent necroptosis. The kinase activity of RIP1 has been associated with a number of inflammatory, neurodegenerative, and oncogenic diseases. In this study, we use the RIP1 kinase inhibitor GNE684 to demonstrate that RIP1 inhibition can effectively block skin inflammation and immune cell infiltrates in livers of Sharpin mutant (Cpdm; chronic proliferative dermatitis) mice in an interventional setting, after disease onset. On the other hand, genetic inactivation of RIP1 (RIP1 KD) or ablation of RIP3 (RIP3 KO) or MLKL (MLKL KO) did not affect testicular pathology of aging male mice. Likewise, infection with vaccinia virus or with mouse gammaherpesvirus MHV68 resulted in similar viral clearance in wild-type, RIP1 KD, and RIP3 KO mice. In summary, this study highlights the benefits of inhibiting RIP1 in skin inflammation, as opposed to its lack of relevance for testicular longevity and the response to certain viral infections.
Asunto(s)
Dermatitis/genética , Infecciones por Herpesviridae/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Piel/inmunología , Vaccinia/genética , Animales , Enfermedad Crónica , Dermatitis/inmunología , Dermatitis/patología , Dermatitis/virología , Modelos Animales de Enfermedad , Gammaherpesvirinae/inmunología , Gammaherpesvirinae/patogenicidad , Regulación de la Expresión Génica , Infecciones por Herpesviridae/patología , Infecciones por Herpesviridae/virología , Inflamación , Hígado/inmunología , Hígado/patología , Hígado/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/deficiencia , Proteínas Quinasas/genética , Proteínas Quinasas/inmunología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunología , Transducción de Señal , Piel/patología , Piel/virología , Testículo/inmunología , Testículo/patología , Testículo/virología , Vaccinia/inmunología , Vaccinia/patología , Vaccinia/virología , Virus Vaccinia/inmunología , Virus Vaccinia/patogenicidad , Replicación Viral/inmunologíaRESUMEN
The kinase RIP1 acts in multiple signaling pathways to regulate inflammatory responses and it can trigger both apoptosis and necroptosis. Its kinase activity has been implicated in a range of inflammatory, neurodegenerative, and oncogenic diseases. Here, we explore the effect of inhibiting RIP1 genetically, using knock-in mice that express catalytically inactive RIP1 D138N, or pharmacologically, using the murine-potent inhibitor GNE684. Inhibition of RIP1 reduced collagen antibody-induced arthritis, and prevented skin inflammation caused by mutation of Sharpin, or colitis caused by deletion of Nemo from intestinal epithelial cells. Conversely, inhibition of RIP1 had no effect on tumor growth or survival in pancreatic tumor models driven by mutant Kras, nor did it reduce lung metastases in a B16 melanoma model. Collectively, our data emphasize a role for the kinase activity of RIP1 in certain inflammatory disease models, but question its relevance to tumor progression and metastases.
Asunto(s)
Inflamación/enzimología , Neoplasias/enzimología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Animales , Artritis/enzimología , Muerte Celular , Línea Celular , Línea Celular Tumoral , Colitis/etiología , Colitis/prevención & control , Dermatitis/enzimología , Femenino , Técnicas de Sustitución del Gen , Humanos , Ileítis/etiología , Ileítis/prevención & control , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Melanoma Experimental/patología , Ratones , Metástasis de la Neoplasia , Neoplasias Pancreáticas/patología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Ratas , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/fisiologíaRESUMEN
Tumor Necrosis Factor alpha (TNFα, TNF) is a key mediator and regulator of mammalian immune responses in healthy organisms and in diseased conditions. TNF governs development of the immune system, cell survival signaling pathways, proliferation and regulates metabolic processes. Whereas TNF-induced NF-κB and MAP pro-survival kinase activities constitute its major biochemical functions, TNF can also stimulate cell death in certain pathological situations. TNF-induced signal transduction pathways are tightly regulated through ubiquitination and phosphorylation of molecules partaking in all TNF-dependent membrane-associated and intracellular protein signaling complexes. Deregulated TNF signaling in individuals carrying naturally occurring genetic mutations in proteins that mediate TNF signaling, or in corresponding genetically modified animal models, results in severe pathologies. In this review we will describe the current knowledge of TNF signaling and its relevance for human health.
Asunto(s)
Citoplasma/inmunología , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Animales , Apoptosis , Muerte Celular/inmunología , Citoplasma/metabolismo , Humanos , Ratones , FN-kappa B/inmunología , FN-kappa B/metabolismo , Fosforilación/inmunología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunología , Receptores Tipo I de Factores de Necrosis Tumoral/inmunología , Transducción de Señal/genética , Factor 2 Asociado a Receptor de TNF/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitinación/inmunologíaRESUMEN
Tumor progression locus 2 (TPL2; also known as MAP3K8) is a mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) that phosphorylates the MAPK kinases MEK1 and MEK2 (MEK1/2), which, in turn, activate the MAPKs extracellular signal-regulated kinase 1 (ERK1) and ERK2 (ERK1/2) in macrophages stimulated through the interleukin-1 receptor (IL-1R), Toll-like receptors (TLRs), or the tumor necrosis factor receptor (TNFR). We describe a conserved and critical role for TPL2 in mediating the effector functions of neutrophils through the activation of the p38 MAPK signaling pathway. Gene expression profiling and functional studies of neutrophils and monocytes revealed a MEK1/2-independent branch point downstream of TPL2 in neutrophils. Biochemical analyses identified the MAPK kinases MEK3 and MEK6 and the MAPKs p38α and p38δ as downstream effectors of TPL2 in these cells. Genetic ablation of the catalytic activity of TPL2 or therapeutic intervention with a TPL2-specific inhibitor reduced the production of inflammatory mediators by neutrophils in response to stimulation with the TLR4 agonist lipopolysaccharide (LPS) in vitro, as well as in rodent models of inflammatory disease. Together, these data suggest that TPL2 is a drug target that activates not only MEK1/2-dependent but also MEK3/6-dependent signaling to promote inflammatory responses.
Asunto(s)
Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Activación Neutrófila , Neutrófilos/enzimología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Activación Enzimática , Inflamación/enzimología , Inflamación/genética , MAP Quinasa Quinasa 3/genética , MAP Quinasa Quinasa 3/metabolismo , MAP Quinasa Quinasa 6/genética , MAP Quinasa Quinasa 6/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Ratones , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Proper regulation of cell death signaling is crucial for the maintenance of homeostasis and prevention of disease. A caspase-independent regulated form of cell death called necroptosis is rapidly emerging as an important mediator of a number of human pathologies including inflammatory bowel disease and ischemia-reperfusion organ injury. Activation of necroptotic signaling through TNF signaling or organ injury leads to the activation of kinases receptor-interacting protein kinases 1 and 3 (RIP1 and RIP3) and culminates in inflammatory cell death. We found that, in addition to phosphorylation, necroptotic cell death is regulated by ubiquitination of RIP1 in the necrosome. Necroptotic RIP1 ubiquitination requires RIP1 kinase activity, but not necroptotic mediators RIP3 and MLKL (mixed lineage kinase-like). Using immunoaffinity enrichment and mass spectrometry, we profiled numerous ubiquitination events on RIP1 that are triggered during necroptotic signaling. Mutation of a necroptosis-related ubiquitination site on RIP1 reduced necroptotic cell death and RIP1 ubiquitination and phosphorylation, and disrupted the assembly of RIP1 and RIP3 in the necrosome, suggesting that necroptotic RIP1 ubiquitination is important for maintaining RIP1 kinase activity in the necrosome complex. We also observed RIP1 ubiquitination in injured kidneys consistent with a physiological role of RIP1 ubiquitination in ischemia-reperfusion disease. Taken together, these data reveal that coordinated and interdependent RIP1 phosphorylation and ubiquitination within the necroptotic complex regulate necroptotic signaling and cell death.
Asunto(s)
Apoptosis , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Apoptosis/efectos de los fármacos , Sistemas CRISPR-Cas/genética , Línea Celular , Creatinina/sangre , Células HEK293 , Células HT29 , Humanos , Enfermedades Renales/etiología , Enfermedades Renales/metabolismo , Ratones , Proteínas de Complejo Poro Nuclear/deficiencia , Proteínas de Complejo Poro Nuclear/genética , Oligopéptidos/farmacología , Fosforilación/efectos de los fármacos , Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Daño por Reperfusión/complicaciones , Daño por Reperfusión/patología , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Ubiquitinación/efectos de los fármacosRESUMEN
Ubiquitination is one of the most prevalent posttranslational modifications in eukaryotic cells, with functional importance in protein degradation, subcellular localization and signal transduction pathways. Immunoaffinity enrichment coupled with quantitative mass spectrometry enables the in-depth characterization of protein ubiquitination events at the site-specific level. We have applied this strategy to investigate cellular response triggered by two distinct type agents: small molecule inhibitors of the tumor-associated kinases MEK and PI3K or the pro-inflammatory cytokine IL-17. Temporal profiling of protein ubiquitination events across a series of time points covering the biological response permits interrogation of signaling through thousands of quantified proteins, of which only a subset display significant and physiologically meaningful regulation. Distinctive clusters of residues within proteins can display distinct temporal patterns attributable to diverse molecular functions, although the majority of differential ubiquitination appears as a coordinated response across the modifiable residues present within an individual substrate. In cells treated with a combination of MEK and PI3K inhibitors, we found differential ubiquitination of MEK within the first hour after treatment and a series of mitochondria proteins at later time points. In the IL-17 signaling pathway, ubiquitination events on several signaling proteins including HOIL-1 and Tollip were observed. The functional relevance of these putative IL-17 mediators was subsequently validated by knockdown of HOIL-1, HOIP and TOLIP, each of which decreased IL-17-stimulated cytokine production. Together, these data validate proteomic profiling of protein ubiquitination as a viable approach for identifying dynamic signaling components in response to intracellular and extracellular perturbations.
Asunto(s)
Proteínas Mitocondriales/metabolismo , Proteínas/inmunología , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Ubiquitina/metabolismo , Apoptosis/efectos de los fármacos , Azetidinas/farmacología , Línea Celular Tumoral/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Indazoles/farmacología , Interleucina-17/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Piperidinas/farmacología , Proteínas/metabolismo , Transducción de Señal , Sulfonamidas/farmacología , Factores de Transcripción , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Precise regulation of survival and signaling pathways is essential for proper maintenance of organismal homeostasis, development, and immune defense. Inhibitor of apoptosis (IAP) proteins are evolutionarily conserved regulators of cell death and immune signaling that impact numerous cellular processes. Initially characterized as inhibitors of apoptosis, the ubiquitin ligase activity of IAP proteins is critical for modulating various signaling pathways (e.g., NF-κB, MAPK) and cellular fate. Cellular IAP1 and IAP2 regulate the pro-survival canonical NF-κB pathway by ubiquitinating RIP1 and enabling recruitment of kinase (IKK) and E3 ligase (LUBAC) complexes. On the other hand, c-IAP1 and c-IAP2 are negative regulators of noncanonical NF-κB signaling by promoting ubiquitination and consequent degradation of the NF-κB-inducing kinase NIK. In this article, we describe the involvement of c-IAP1 and c-IAP2 in NF-κB signaling and provide detailed methodology for examining how c-IAPs exert their functional roles.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/metabolismo , FN-kappa B/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal , Western Blotting/métodos , Línea Celular , Activación Enzimática , Expresión Génica , Humanos , Inmunoprecipitación/métodos , Ligandos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , ARN Interferente Pequeño/genética , Receptores del Factor de Necrosis Tumoral/agonistas , Fracciones Subcelulares , TransfecciónRESUMEN
Evasion of cell death is one crucial capability acquired by tumour cells to ward-off anti-tumour therapies and represents a fundamental challenge to sustaining clinical efficacy for currently available agents. Inhibitor of apoptosis (IAP) proteins use their ubiquitin E3 ligase activity to promote cancer cell survival by mediating proliferative signalling and blocking cell death in response to diverse stimuli. Using immunoaffinity enrichment and MS, ubiquitination sites on thousands of proteins were profiled upon initiation of cell death by IAP antagonists in IAP antagonist-sensitive and -resistant breast cancer cell lines. Our analyses identified hundreds of proteins with elevated levels of ubiquitin-remnant [K-GG (Lys-Gly-Gly)] peptides upon activation of cell death by the IAP antagonist BV6. The majority of these were observed in BV6-sensitive, but not-resistant, cells. Among these were known pro-apoptotic regulators, including CYC (cytochrome c), RIP1 (receptor-interacting protein 1) and a selection of proteins known to reside in the mitochondria or regulate NF-κB (nuclear factor κB) signalling. Analysis of early time-points revealed that IAP antagonist treatment stimulated rapid ubiquitination of NF-κB signalling proteins, including TRAF2 [TNF (tumour necrosis factor) receptor-associated factor 2], HOIL-1 (haem-oxidized iron-regulatory protein 2 ubiquitin ligase-1), NEMO (NF-κB essential modifier), as well as c-IAP1 (cellular IAP1) auto-ubiquitination. Knockdown of several NF-κB pathway members reduced BV6-induced cell death and TNF production in sensitive cell lines. Importantly, RIP1 was found to be constitutively ubiquitinated in sensitive breast-cancer cell lines at higher basal level than in resistant cell lines. Together, these data show the diverse and temporally defined roles of protein ubiquitination following IAP-antagonist treatment and provide critical insights into predictive diagnostics that may enhance clinical efficacy.
Asunto(s)
Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Inhibidoras de la Apoptosis/genética , Oligopéptidos/farmacología , Ubiquitina/genética , Línea Celular Tumoral , Citocromos c/genética , Citocromos c/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Perfilación de la Expresión Génica , Humanos , Quinasa I-kappa B/antagonistas & inhibidores , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Proteínas Inhibidoras de la Apoptosis/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Proteolisis , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Factor 2 Asociado a Receptor de TNF/genética , Factor 2 Asociado a Receptor de TNF/metabolismo , Factores de Transcripción , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
The cellular inhibitor of apoptosis (c-IAP) proteins are E3 ubiquitin ligases that are critical regulators of tumour necrosis factor (TNF) receptor (TNFR)-mediated signalling. Through their E3 ligase activity c-IAP proteins promote ubiquitination of receptor-interaction protein 1 (RIP1), NF-κB-inducing kinase (NIK) and themselves, and regulate the assembly of TNFR signalling complexes. Consequently, in the absence of c-IAP proteins, TNFR-mediated activation of NF-κB and MAPK pathways and the induction of gene expression are severely reduced. Here, we describe the identification of OTUB1 as a c-IAP-associated deubiquitinating enzyme that regulates c-IAP1 stability. OTUB1 disassembles K48-linked polyubiquitin chains from c-IAP1 in vitro and in vivo within the TWEAK receptor-signalling complex. Downregulation of OTUB1 promotes TWEAK- and IAP antagonist-stimulated caspase activation and cell death, and enhances c-IAP1 degradation. Furthermore, knockdown of OTUB1 reduces TWEAK-induced activation of canonical NF-κB and MAPK signalling pathways and modulates TWEAK-induced gene expression. Finally, suppression of OTUB1 expression in zebrafish destabilizes c-IAP (Birc2) protein levels and disrupts fish vasculature. These results suggest that OTUB1 regulates NF-κB and MAPK signalling pathways and TNF-dependent cell death by modulating c-IAP1 stability.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Transducción de Señal , Ubiquitina/metabolismo , Animales , Vasos Sanguíneos/embriología , Línea Celular , Enzimas Desubicuitinizantes , Humanos , Hidrólisis , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Pez Cebra/embriologíaRESUMEN
ML-IAP [melanoma IAP (inhibitor of apoptosis)] is an anti-apoptotic protein that is expressed highly in melanomas where it contributes to resistance to apoptotic stimuli. The anti-apoptotic activity and elevated expression of IAP family proteins in many human cancers makes IAP proteins attractive targets for inhibition by cancer therapeutics. Small-molecule IAP antagonists that bind with high affinities to select BIR (baculovirus IAP repeat) domains have been shown to stimulate auto-ubiquitination and rapid proteasomal degradation of c-IAP1 (cellular IAP1) and c-IAP2 (cellular IAP2). In the present paper, we report ML-IAP proteasomal degradation in response to bivalent, but not monovalent, IAP antagonists. This degradation required ML-IAP ubiquitin ligase activity and was independent of c-IAP1 or c-IAP2. Although ML-IAP is best characterized in melanoma cells, we show that ML-IAP expression in normal mammalian tissues is restricted largely to the eye, being most abundant in ciliary body epithelium and retinal pigment epithelium. Surprisingly, given this pattern of expression, gene-targeted mice lacking ML-IAP exhibited normal intraocular pressure as well as normal retinal structure and function. The results of the present study indicate that ML-IAP is dispensable for both normal mouse development and ocular homoeostasis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Ojo/metabolismo , Proteínas Inhibidoras de la Apoptosis/fisiología , Proteínas de Neoplasias/fisiología , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Línea Celular Tumoral , Ojo/irrigación sanguínea , Femenino , Humanos , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Proteínas Inhibidoras de la Apoptosis/genética , Presión Intraocular , Masculino , Melanoma , Ratones , Ratones Mutantes , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Especificidad de Órganos , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Estructura Terciaria de Proteína , Retina/anatomía & histología , Retina/fisiología , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
A series of compounds were designed and synthesized as antagonists of cIAP1/2, ML-IAP, and XIAP based on the N-terminus, AVPI, of mature Smac. Compound 1 (GDC-0152) has the best profile of these compounds; it binds to the XIAP BIR3 domain, the BIR domain of ML-IAP, and the BIR3 domains of cIAP1 and cIAP2 with K(i) values of 28, 14, 17, and 43 nM, respectively. These compounds promote degradation of cIAP1, induce activation of caspase-3/7, and lead to decreased viability of breast cancer cells without affecting normal mammary epithelial cells. Compound 1 inhibits tumor growth when dosed orally in the MDA-MB-231 breast cancer xenograft model. Compound 1 was advanced to human clinical trials, and it exhibited linear pharmacokinetics over the dose range (0.049 to 1.48 mg/kg) tested. Mean plasma clearance in humans was 9 ± 3 mL/min/kg, and the volume of distribution was 0.6 ± 0.2 L/kg.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Tiadiazoles/síntesis química , Tiadiazoles/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Apoptosis/efectos de los fármacos , Proteína 3 que Contiene Repeticiones IAP de Baculovirus , Unión Competitiva , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Caspasas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ensayos Clínicos Fase I como Asunto , Femenino , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Masculino , Tiadiazoles/química , Tiadiazoles/farmacocinética , Ubiquitina-Proteína LigasasRESUMEN
Tumor necrosis factor (TNF) family members are essential for the development and proper functioning of the immune system. TNF receptor (TNFR) signaling is mediated through the assembly of protein signaling complexes that activate the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways in a ubiquitin-dependent manner. The cellular inhibitor of apoptosis (c-IAP) proteins c-IAP1 and c-IAP2 are E3 ubiquitin ligases that are recruited to TNFR signaling complexes through their constitutive association with the adaptor protein TNFR-associated factor 2 (TRAF2). We demonstrated that c-IAP1 and c-IAP2 were required for canonical activation of NF-κB and MAPK by members of the TNFR family. c-IAPs were required for the recruitment of inhibitor of κB kinase ß (IKKß), the IKK regulatory subunit NF-κB essential modulator (NEMO), and RBCK1/Hoil1-interacting protein (HOIP) to TNFR signaling complexes and the induction of gene expression by TNF family members. In contrast, TNFRs that stimulated the noncanonical NF-κB pathway triggered translocation of c-IAPs, TRAF2, and TRAF3 from the cytosol to membrane fractions, which led to their proteasomal and lysosomal degradation. Finally, we established that signaling by B cell-activating factor receptor 3 induced the cytosolic depletion of TRAF3, which enabled noncanonical NF-κB activation. These results define c-IAP proteins as critical regulators of the activation of NF-κB and MAPK signaling pathways by members of the TNFR superfamily.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Transducción de Señal/inmunología , Factores de Necrosis Tumoral/metabolismo , Western Blotting , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Silenciador del Gen , Humanos , Quinasa I-kappa B/metabolismo , Proteínas Inhibidoras de la Apoptosis/inmunología , Transporte de Proteínas , ARN Interferente Pequeño/genética , Receptores de Interleucina-4/metabolismo , Factor 2 Asociado a Receptor de TNF/metabolismo , Factores de Necrosis Tumoral/inmunología , Ubiquitina/metabolismo , Ubiquitina-Proteína LigasasRESUMEN
Inhibitor of apoptosis (IAP) proteins are negative regulators of cell death. IAP family members contain RING domains that impart E3 ubiquitin ligase activity. Binding of endogenous or small-molecule antagonists to select baculovirus IAP repeat (BIR) domains within cellular IAP (cIAP) proteins promotes autoubiquitination and proteasomal degradation and so releases inhibition of apoptosis mediated by cIAP. Although the molecular details of antagonist-BIR domain interactions are well understood, it is not clear how this binding event influences the activity of the RING domain. Here biochemical and structural studies reveal that the unliganded, multidomain cIAP1 sequesters the RING domain within a compact, monomeric structure that prevents RING dimerization. Antagonist binding induces conformational rearrangements that enable RING dimerization and formation of the active E3 ligase.