RESUMEN
Necroptosis is a lytic form of cell death that is mediated by the kinase RIPK3 and the pseudokinase MLKL when caspase-8 is inhibited downstream of death receptors, toll-like receptor 3 (TLR3), TLR4, and the intracellular Z-form nucleic acid sensor ZBP1. Oligomerization and activation of RIPK3 is driven by interactions with the kinase RIPK1, the TLR adaptor TRIF, or ZBP1. In this study, we use immunohistochemistry (IHC) and in situ hybridization (ISH) assays to generate a tissue atlas characterizing RIPK1, RIPK3, Mlkl, and ZBP1 expression in mouse tissues. RIPK1, RIPK3, and Mlkl were co-expressed in most immune cell populations, endothelial cells, and many barrier epithelia. ZBP1 was expressed in many immune populations, but had more variable expression in epithelia compared to RIPK1, RIPK3, and Mlkl. Intriguingly, expression of ZBP1 was elevated in Casp8-/- Tnfr1-/- embryos prior to their succumbing to aberrant necroptosis around embryonic day 15 (E15). ZBP1 contributed to this embryonic lethality because rare Casp8-/- Tnfr1-/- Zbp1-/- mice survived until after birth. Necroptosis mediated by TRIF contributed to the demise of Casp8-/- Tnfr1-/- Zbp1-/- pups in the perinatal period. Of note, Casp8-/- Tnfr1-/- Trif-/- Zbp1-/- mice exhibited autoinflammation and morbidity, typically within 5-7 weeks of being born, which is not seen in Casp8-/- Ripk1-/- Trif-/- Zbp1-/-, Casp8-/- Ripk3-/-, or Casp8-/- Mlkl-/- mice. Therefore, after birth, loss of caspase-8 probably unleashes RIPK1-dependent necroptosis driven by death receptors other than TNFR1.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular , Caspasa 8 , Ratones Noqueados , Necroptosis , Proteínas de Unión al ARN , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Receptores Tipo I de Factores de Necrosis Tumoral , Animales , Ratones , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Caspasa 8/metabolismo , Caspasa 8/genética , Ratones Endogámicos C57BL , Proteínas Quinasas/metabolismo , Proteínas Quinasas/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
The proteolytic activity of caspase-8 suppresses lethal RIPK1-, RIPK3- and MLKL-dependent necroptosis during mouse embryogenesis. Caspase-8 is reported to cleave RIPK3 in addition to the RIPK3-interacting kinase RIPK1, but whether cleavage of RIPK3 is crucial for necroptosis suppression is unclear. Here we show that caspase-8-driven cleavage of endogenous mouse RIPK3 after Asp333 is dependent on downstream caspase-3. Consistent with RIPK3 cleavage being a consequence of apoptosis rather than a critical brake on necroptosis, Ripk3D333A/D333A knock-in mice lacking the Asp333 cleavage site are viable and develop normally. Moreover, in contrast to mice lacking caspase-8 in their intestinal epithelial cells, Ripk3D333A/D333A mice do not exhibit increased sensitivity to high dose tumor necrosis factor (TNF). Ripk3D333A/D333A macrophages died at the same rate as wild-type (WT) macrophages in response to TNF plus cycloheximide, TNF plus emricasan, or infection with murine cytomegalovirus (MCMV) lacking M36 and M45 to inhibit caspase-8 and RIPK3 activation, respectively. We conclude that caspase cleavage of RIPK3 is dispensable for mouse development, and that cleavage of caspase-8 substrates, including RIPK1, is sufficient to prevent necroptosis.
Asunto(s)
Caspasas , Proteínas Quinasas , Animales , Ratones , Apoptosis , Caspasa 8/genética , Caspasa 8/metabolismo , Desarrollo Embrionario , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Caspase-8 is a protease with both pro-death and pro-survival functions: it mediates apoptosis induced by death receptors such as TNFR11, and suppresses necroptosis mediated by the kinase RIPK3 and the pseudokinase MLKL2-4. Mice that lack caspase-8 display MLKL-dependent embryonic lethality4, as do mice that express catalytically inactive CASP8(C362A)5. Casp8C362A/C362AMlkl-/- mice die during the perinatal period5, whereas Casp8-/-Mlkl-/- mice are viable4, which indicates that inactive caspase-8 also has a pro-death scaffolding function. Here we show that mutant CASP8(C362A) induces the formation of ASC (also known as PYCARD) specks, and caspase-1-dependent cleavage of GSDMD and caspases 3 and 7 in MLKL-deficient mouse intestines around embryonic day 18. Caspase-1 and its adaptor ASC contributed to the perinatal lethal phenotype because a number of Casp8C362A/C362AMlkl-/-Casp1-/- and Casp8C362A/C362AMlkl-/-Asc-/- mice survived beyond weaning. Transfection studies suggest that inactive caspase-8 adopts a distinct conformation to active caspase-8, enabling its prodomain to engage ASC. Upregulation of the lipopolysaccharide sensor caspase-11 in the intestines of both Casp8C362A/C362AMlkl-/- and Casp8C362A/C362AMlkl-/-Casp1-/- mice also contributed to lethality because Casp8C362A/C362AMlkl-/-Casp1-/-Casp11-/- (Casp11 is also known as Casp4) neonates survived more often than Casp8C362A/C362AMlkl-/-Casp1-/- neonates. Finally, Casp8C362A/C362ARipk3-/-Casp1-/-Casp11-/- mice survived longer than Casp8C362A/C362AMlkl-/-Casp1-/-Casp11-/- mice, indicating that a necroptosis-independent function of RIPK3 also contributes to lethality. Thus, unanticipated plasticity in death pathways is revealed when caspase-8-dependent apoptosis and MLKL-dependent necroptosis are inhibited.
Asunto(s)
Caspasa 8/metabolismo , Muerte Celular/genética , Mucosa Intestinal/citología , Animales , Proteínas Adaptadoras de Señalización CARD/metabolismo , Caspasa 8/genética , Femenino , Regulación de la Expresión Génica , Células HEK293 , Humanos , Mucosa Intestinal/enzimología , Estimación de Kaplan-Meier , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismoRESUMEN
The aspartate-specific cysteine protease caspase-8 suppresses necroptotic cell death mediated by RIPK3 and MLKL. Indeed, mice that lack caspase-8 die in a RIPK3- and MLKL-dependent manner during embryogenesis1-3. In humans, caspase-8 deficiency is associated with immunodeficiency4 or very early onset inflammatory bowel disease5. The substrates that are cleaved by caspase-8 to prevent necroptosis in vivo have not been defined. Here we show that knock-in mice that express catalytically inactive caspase-8(C362A) die as embryos owing to MLKL-dependent necroptosis, similar to caspase-8-deficient mice. Thus, caspase-8 must cleave itself, other proteins or both to inhibit necroptosis. Mice that express caspase-8(D212A/D218A/D225A/D387A), which cannot cleave itself, were viable, as were mice that express c-FLIP or CYLD proteins that had been mutated to prevent cleavage by caspase-8. By contrast, mice that express RIPK1(D325A), in which the caspase-8 cleavage site Asp325 had been mutated, died mid-gestation. Embryonic lethality was prevented by inactivation of RIPK1, loss of TNFR1, or loss of both MLKL and the caspase-8 adaptor FADD, but not by loss of MLKL alone. Thus, RIPK1(D325A) appears to trigger cell death mediated by TNF, the kinase activity of RIPK1 and FADD-caspase-8. Accordingly, dying endothelial cells that contain cleaved caspase-3 were abnormally abundant in yolk sacs of Ripk1D325A/D325A embryos. Heterozygous Ripk1D325A/+ cells and mice were viable, but were also more susceptible to TNF-induced cell death than were wild-type cells or mice. Our data show that Asp325 of RIPK1 is essential for limiting aberrant cell death in response to TNF, consistent with the idea that cleavage of RIPK1 by caspase-8 is a mechanism for dismantling death-inducing complexes.
Asunto(s)
Apoptosis/fisiología , Caspasa 8/metabolismo , Necroptosis/fisiología , Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Desarrollo Embrionario/genética , Humanos , Ratones , Mutación , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genéticaRESUMEN
OTULIN (OTU deubiquitinase with linear linkage specificity) removes linear polyubiquitin from proteins that have been modified by LUBAC (linear ubiquitin chain assembly complex) and is critical for preventing auto-inflammatory disease1,2 and embryonic lethality during mouse development3. Here we show that OTULIN promotes rather than counteracts LUBAC activity by preventing its auto-ubiquitination with linear polyubiquitin. Thus, knock-in mice that express catalytically inactive OTULIN, either constitutively or selectively in endothelial cells, resembled LUBAC-deficient mice4 and died midgestation as a result of cell death mediated by TNFR1 (tumour necrosis factor receptor 1) and the kinase activity of RIPK1 (receptor-interacting protein kinase 1). Inactivation of OTULIN in adult mice also caused pro-inflammatory cell death. Accordingly, embryonic lethality and adult auto-inflammation were prevented by the combined loss of cell death mediators: caspase 8 for apoptosis and RIPK3 for necroptosis. Unexpectedly, OTULIN mutant mice that lacked caspase 8 and RIPK3 died in the perinatal period, exhibiting enhanced production of type I interferon that was dependent on RIPK1. Collectively, our results indicate that OTULIN and LUBAC function in a linear pathway, and highlight a previously unrecognized interaction between linear ubiquitination, regulators of cell death, and induction of type I interferon.
Asunto(s)
Muerte Celular , Enzimas Desubicuitinizantes/metabolismo , Endopeptidasas/metabolismo , Inflamación/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitinación , Animales , Caspasa 8/genética , Caspasa 8/metabolismo , Muerte Celular/genética , Enzimas Desubicuitinizantes/genética , Pérdida del Embrión/genética , Endopeptidasas/genética , Inflamación/enzimología , Inflamación/genética , Interferón Tipo I/biosíntesis , Ratones , Ratones Endogámicos C57BL , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Ubiquitinación/genética , Pérdida de Peso/genéticaRESUMEN
The necroptotic cell death pathway is a key component of human pathogen defense that can become aberrantly derepressed during tissue homeostasis to contribute to multiple types of tissue damage and disease. While formation of the necrosome kinase signaling complex containing RIPK1, RIPK3, and MLKL has been extensively characterized, additional mechanisms of its regulation and effector functions likely remain to be discovered. We screened 19,883 mouse protein-coding genes by CRISPR/Cas9-mediated gene knockout for resistance to cytokine-induced necroptosis and identified 112 regulators and mediators of necroptosis, including 59 new candidate pathway components with minimal or no effect on cell growth in the absence of necroptosis induction. Among these, we further characterized the function of PTBP1, an RNA binding protein whose activity is required to maintain RIPK1 protein abundance by regulating alternative splice-site selection.
Asunto(s)
Empalme Alternativo , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Fibroblastos/enzimología , Marcación de Gen/métodos , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Necroptosis , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Fibroblastos/patología , Regulación Enzimológica de la Expresión Génica , Células HEK293 , Células HT29 , Ribonucleoproteínas Nucleares Heterogéneas/genética , Humanos , Ratones , Proteína de Unión al Tracto de Polipirimidina/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Transducción de SeñalRESUMEN
Receptor-interacting protein kinase 1 (RIPK1) promotes cell survival-mice lacking RIPK1 die perinatally, exhibiting aberrant caspase-8-dependent apoptosis and mixed lineage kinase-like (MLKL)-dependent necroptosis. However, mice expressing catalytically inactive RIPK1 are viable, and an ill-defined pro-survival function for the RIPK1 scaffold has therefore been proposed. Here we show that the RIP homotypic interaction motif (RHIM) in RIPK1 prevents the RHIM-containing adaptor protein ZBP1 (Z-DNA binding protein 1; also known as DAI or DLM1) from activating RIPK3 upstream of MLKL. Ripk1RHIM/RHIM mice that expressed mutant RIPK1 with critical RHIM residues IQIG mutated to AAAA died around birth and exhibited RIPK3 autophosphorylation on Thr231 and Ser232, which is a hallmark of necroptosis, in the skin and thymus. Blocking necroptosis with catalytically inactive RIPK3(D161N), RHIM mutant RIPK3, RIPK3 deficiency, or MLKL deficiency prevented lethality in Ripk1RHIM/RHIM mice. Loss of ZBP1, which engages RIPK3 in response to certain viruses but previously had no defined role in development, also prevented perinatal lethality in Ripk1RHIM/RHIM mice. Consistent with the RHIM of RIPK1 functioning as a brake that prevents ZBP1 from engaging the RIPK3 RHIM, ZBP1 interacted with RIPK3 in Ripk1RHIM/RHIMMlkl-/- macrophages, but not in wild-type, Mlkl-/- or Ripk1RHIM/RHIMRipk3RHIM/RHIM macrophages. Collectively, these findings indicate that the RHIM of RIPK1 is critical for preventing ZBP1/RIPK3/MLKL-dependent necroptosis during development.
Asunto(s)
Apoptosis , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Glicoproteínas/antagonistas & inhibidores , Glicoproteínas/metabolismo , Necrosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/deficiencia , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Secuencias de Aminoácidos , Animales , Animales Recién Nacidos , Caspasa 8/genética , Caspasa 8/metabolismo , Embrión de Mamíferos/citología , Femenino , Glicoproteínas/química , Glicoproteínas/deficiencia , Macrófagos/metabolismo , Masculino , Ratones , Mutación , Fosforilación , Unión Proteica , Proteínas Quinasas/deficiencia , Proteínas Quinasas/metabolismo , Proteínas de Unión al ARN , Proteína Serina-Treonina Quinasas de Interacción con Receptores/química , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Protein modification with ubiquitin chains is an essential signaling event catalyzed by E3 ubiquitin ligases. Most human E3s contain a signature RING domain that recruits a ubiquitin-charged E2 and a separate domain for substrate recognition. How RING-E3s can build polymeric ubiquitin chains while binding substrates and E2s at defined interfaces remains poorly understood. Here, we show that the RING-E3 APC/C catalyzes chain elongation by strongly increasing the affinity of its E2 for the distal acceptor ubiquitin in a growing conjugate. This function of the APC/C requires its coactivator as well as conserved residues of the E2 and ubiquitin. APC/C's ability to track the tip of an emerging conjugate is required for APC/C-substrate degradation and accurate cell division. Our results suggest that RING-E3s tether the distal ubiquitin of a growing chain in proximity to the active site of their E2s, allowing them to assemble polymeric conjugates without altering their binding to substrate or E2.
Asunto(s)
Subunidad Apc11 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Subunidad Apc2 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas Cdc20/metabolismo , Biosíntesis de Péptidos Independientes de Ácidos Nucleicos/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina/genética , Dominio Catalítico , Proteínas Cdc20/genética , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Activación Enzimática , Células HeLa , Humanos , Unión Proteica , Estructura Terciaria de Proteína , Interferencia de ARN , ARN Interferente Pequeño , Ubiquitina/biosíntesis , UbiquitinaciónRESUMEN
Receptor-interacting protein kinase 1 (RIPK1) and RIPK3 trigger pro-inflammatory cell death termed "necroptosis." Studies with RIPK3-deficient mice or the RIPK1 inhibitor necrostatin-1 suggest that necroptosis exacerbates pathology in many disease models. We engineered mice expressing catalytically inactive RIPK3 D161N or RIPK1 D138N to determine the need for the active kinase in the whole animal. Unexpectedly, RIPK3 D161N promoted lethal RIPK1- and caspase-8-dependent apoptosis. In contrast, mice expressing RIPK1 D138N were viable and, like RIPK3-deficient mice, resistant to tumor necrosis factor (TNF)-induced hypothermia. Cells expressing RIPK1 D138N were resistant to TNF-induced necroptosis, whereas TNF-induced signaling pathways promoting gene transcription were unperturbed. Our data indicate that the kinase activity of RIPK3 is essential for necroptosis but also governs whether a cell activates caspase-8 and dies by apoptosis.
Asunto(s)
Apoptosis , Necrosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Caspasa 8/genética , Caspasa 8/metabolismo , Supervivencia Celular , Pérdida del Embrión , Desarrollo Embrionario , Enteritis/patología , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Técnicas de Sustitución del Gen , Intestino Grueso/patología , Intestino Delgado/patología , Ratones , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Antibodies that specifically recognize polyubiquitin chains containing ubiquitins linked at a particular lysine residue are powerful tools for interrogating endogenous protein modifications. Here, we describe protocols for revealing K11-, K48-, and K63-linked polyubiquitin chains by western blotting, immunoprecipitation, or immunostaining.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Poliubiquitina/química , Poliubiquitina/inmunología , Western Blotting/métodos , Inmunoprecipitación/métodos , Poliubiquitina/metabolismo , Procesamiento Proteico-Postraduccional , Enzimas Activadoras de Ubiquitina , Enzimas Ubiquitina-Conjugadoras , Ubiquitina-Proteína Ligasas , UbiquitinaciónRESUMEN
Packaging of proteins from the endoplasmic reticulum into COPII vesicles is essential for secretion. In cells, most COPII vesicles are approximately 60-80 nm in diameter, yet some must increase their size to accommodate 300-400 nm procollagen fibres or chylomicrons. Impaired COPII function results in collagen deposition defects, cranio-lenticulo-sutural dysplasia, or chylomicron retention disease, but mechanisms to enlarge COPII coats have remained elusive. Here, we identified the ubiquitin ligase CUL3-KLHL12 as a regulator of COPII coat formation. CUL3-KLHL12 catalyses the monoubiquitylation of the COPII-component SEC31 and drives the assembly of large COPII coats. As a result, ubiquitylation by CUL3-KLHL12 is essential for collagen export, yet less important for the transport of small cargo. We conclude that monoubiquitylation controls the size and function of a vesicle coat.
Asunto(s)
Vesículas Cubiertas por Proteínas de Revestimiento/química , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Ubiquitina/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Línea Celular , Forma de la Célula , Colágeno/metabolismo , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células HeLa , Humanos , Ratones , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Transporte de Proteínas , Ubiquitinación , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Modification of proteins with ubiquitin chains is an essential regulatory event in cell cycle control. Differences in the connectivity of ubiquitin chains are believed to result in distinct functional consequences for the modified proteins. Among eight possible homogenous chain types, canonical Lys48-linked ubiquitin chains have long been recognized to drive the proteasomal degradation of cell cycle regulators, and Lys48 is the only essential lysine residue of ubiquitin in yeast. It thus came as a surprise that in higher eukaryotes atypical K11-linked ubiquitin chains regulate the substrates of the anaphase-promoting complex and control progression through mitosis. We discuss recent findings that shed light on the assembly and function of K11-linked chains during cell division.
Asunto(s)
División Celular , Poliubiquitina/fisiología , Ciclosoma-Complejo Promotor de la Anafase , Animales , Humanos , Multimerización de Proteína , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Proteínas Ubiquitinadas/metabolismo , UbiquitinaciónRESUMEN
Ubiquitin chains of different topologies trigger distinct functional consequences, including protein degradation and reorganization of complexes. The assembly of most ubiquitin chains is promoted by E2s, yet how these enzymes achieve linkage specificity is poorly understood. We have discovered that the K11-specific Ube2S orients the donor ubiquitin through an essential noncovalent interaction that occurs in addition to the thioester bond at the E2 active site. The E2-donor ubiquitin complex transiently recognizes the acceptor ubiquitin, primarily through electrostatic interactions. The recognition of the acceptor ubiquitin surface around Lys11, but not around other lysines, generates a catalytically competent active site, which is composed of residues of both Ube2S and ubiquitin. Our studies suggest that monomeric E2s promote linkage-specific ubiquitin chain formation through substrate-assisted catalysis.
Asunto(s)
Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina/metabolismo , Catálisis , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Ubiquitina/química , Enzimas Ubiquitina-Conjugadoras/químicaRESUMEN
Polyubiquitination is a posttranslational modification where ubiquitin chains containing isopeptide bonds linking one of seven ubiquitin lysines with the C terminus of an adjoining ubiquitin are covalently attached to proteins. While functions of K48- and K63-linked polyubiquitin are understood, the role(s) of noncanonical K11-linked chains is less clear. A crystal structure of K11-linked diubiquitin demonstrates a distinct conformation from K48- or K63-linked diubiquitin. We engineered a K11 linkage-specific antibody and use it to demonstrate that K11 chains are highly upregulated in mitotic human cells precisely when substrates of the ubiquitin ligase anaphase-promoting complex (APC/C) are degraded. These chains increased with proteasomal inhibition, suggesting they act as degradation signals in vivo. Inhibition of the APC/C strongly impeded the formation of K11-linked chains, suggesting that a single ubiquitin ligase is the major source of mitotic K11-linked chains. Our results underscore the importance of K11-linked ubiquitin chains as critical regulators of mitotic protein degradation.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Ciclo Celular/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina/metabolismo , Ubiquitinación/fisiología , Ciclosoma-Complejo Promotor de la Anafase , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Células HeLa , Humanos , Ubiquitina/química , Ubiquitina/inmunologíaRESUMEN
Ubiquitination by the anaphase-promoting complex (APC/C) is essential for proliferation in all eukaryotes. The human APC/C promotes the degradation of mitotic regulators by assembling K11-linked ubiquitin chains, the formation of which is initiated by its E2 UbcH10. Here, we identify the conserved Ube2S as a K11-specific chain elongating E2 for human and Drosophila APC/C. Ube2S depends on the cell cycle-dependent association with the APC/C activators Cdc20 and Cdh1 for its activity. While depletion of Ube2S already inhibits APC/C in cells, the loss of the complete UbcH10/Ube2S-module leads to dramatic stabilization of APC/C substrates, severe spindle defects, and a strong mitotic delay. Ube2S and UbcH10 are tightly co-regulated in the cell cycle by APC/C-dependent degradation. We conclude that UbcH10 and Ube2S constitute a physiological E2-module for APC/C, the activity of which is required for spindle assembly and cell division.
Asunto(s)
Drosophila melanogaster/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ciclosoma-Complejo Promotor de la Anafase , Animales , Biocatálisis , Células HeLa , Humanos , Mitosis , Unión Proteica , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genéticaRESUMEN
Mammalian Toll-like receptors (TLRs) 3, 7, 8 and 9 initiate immune responses to infection by recognizing microbial nucleic acids; however, these responses come at the cost of potential autoimmunity owing to inappropriate recognition of self nucleic acids. The localization of TLR9 and TLR7 to intracellular compartments seems to have a role in facilitating responses to viral nucleic acids while maintaining tolerance to self nucleic acids, yet the cell biology regulating the transport and localization of these receptors remains poorly understood. Here we define the route by which TLR9 and TLR7 exit the endoplasmic reticulum and travel to endolysosomes in mouse macrophages and dendritic cells. The ectodomains of TLR9 and TLR7 are cleaved in the endolysosome, such that no full-length protein is detectable in the compartment where ligand is recognized. Notably, although both the full-length and cleaved forms of TLR9 are capable of binding ligand, only the processed form recruits MyD88 on activation, indicating that this truncated receptor, rather than the full-length form, is functional. Furthermore, conditions that prevent receptor proteolysis, including forced TLR9 surface localization, render the receptor non-functional. We propose that ectodomain cleavage represents a strategy to restrict receptor activation to endolysosomal compartments and prevent TLRs from responding to self nucleic acids.
Asunto(s)
Procesamiento Proteico-Postraduccional , Receptor Toll-Like 9/química , Receptor Toll-Like 9/metabolismo , Animales , Línea Celular , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/metabolismo , Retículo Endoplásmico/metabolismo , Femenino , Aparato de Golgi/metabolismo , Ligandos , Lisosomas/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Ratones , Factor 88 de Diferenciación Mieloide/metabolismo , Fagosomas/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas , Receptor Toll-Like 7/química , Receptor Toll-Like 7/metabolismoRESUMEN
Anthrax lethal toxin (LT) rapidly kills macrophages from certain mouse strains in a mechanism dependent on the breakdown of unknown protein(s) by the proteasome, formation of the Nalp1b (NLRP1b) inflammasome and subsequent activation of caspase-1. We report that heat-shocking LT-sensitive macrophages rapidly protects them against cytolysis by inhibiting caspase-1 activation without upstream effects on LT endocytosis or cleavage of the toxin's known cytosolic substrates (mitogen-activated protein kinases). Heat shock protection against LT occurred through a mechanism independent of de novo protein synthesis, HSP90 activity, p38 activation or proteasome inhibition and was downstream of mitogen-activated protein kinase cleavage and degradation of an unknown substrate by the proteasome. The heat shock inhibition of LT-mediated caspase-1 activation was not specific to the Nalp1b (NLRP1b) inflammasome, as heat shock also inhibited Nalp3 (NLRP3) inflammasome-mediated caspase-1 activation in macrophages. We found that heat shock induced pro-caspase-1 association with a large cellular complex that could prevent its activation. Additionally, while heat-shocking recombinant caspase-1 did not affect its activity in vitro, lysates from heat-shocked cells completely inhibited recombinant active caspase-1 activity. Our results suggest that heat shock inhibition of active caspase-1 can occur independently of an inflammasome platform, through a titratable factor present within intact, functioning heat-shocked cells.
Asunto(s)
Antígenos Bacterianos/toxicidad , Toxinas Bacterianas/toxicidad , Caspasa 1/metabolismo , Inhibidores de Caspasas , Calor , Macrófagos/enzimología , Macrófagos/efectos de la radiación , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular , Supervivencia Celular , Ratones , Proteína con Dominio Pirina 3 de la Familia NLRRESUMEN
Macrophages from certain inbred mouse strains are rapidly killed (< 90 min) by anthrax lethal toxin (LT). LT cleaves cytoplasmic MEK proteins at 20 min and induces caspase-1 activation in sensitive macrophages at 50-60 min, but the mechanism of LT-induced death is unknown. Proteasome inhibitors block LT-mediated caspase-1 activation and can protect against cell death, indicating that the degradation of at least one cellular protein is required for LT-mediated cell death. Proteins can be degraded by the proteasome via the N-end rule, in which a protein's stability is determined by its N-terminal residue. Using amino acid derivatives that act as inhibitors of this pathway, we show that the N-end rule is required for LT-mediated caspase-1 activation and cell death. We also found that bestatin methyl ester, an aminopeptidase inhibitor protects against LT in vitro and in vivo and that the different inhibitors of the protein degradation pathway act synergistically in protecting against LT. We identify c-IAP1, a mammalian member of the inhibitor of apoptosis protein (IAP) family, as a novel N-end rule substrate degraded in macrophages treated with LT. We also show that LT-induced c-IAP1 degradation is independent of the IAP-antagonizing proteins Smac/DIABLO and Omi/HtrA2, but dependent on caspases.
Asunto(s)
Antígenos Bacterianos/metabolismo , Bacillus anthracis/enzimología , Toxinas Bacterianas/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Macrófagos/metabolismo , Metaloendopeptidasas/metabolismo , Aminopeptidasas/antagonistas & inhibidores , Aminopeptidasas/farmacología , Animales , Carbunco/microbiología , Bacillus anthracis/patogenicidad , Caspasa 1/metabolismo , Muerte Celular , Células Cultivadas , Proteínas Inhibidoras de la Apoptosis/aislamiento & purificación , Leucina/análogos & derivados , Leucina/farmacología , Macrófagos/patología , Redes y Vías Metabólicas , Ratones , Ratones Endogámicos BALB C , Especificidad por Sustrato , VirulenciaRESUMEN
Anthrax lethal toxin (LT) is cytotoxic to macrophages from certain inbred mouse strains. The gene controlling macrophage susceptibility to LT is Nalp1b. Nalp1b forms part of the inflammasome, a multiprotein complex involved in caspase-1 activation and release of interleukin (IL)-1beta and IL-18. We confirm the role of caspase-1 in LT-mediated death by showing that caspase inhibitors differentially protected cells against LT, with the degree of protection corresponding to each compound's ability to inhibit caspase-1. Caspase-1 activation and cytokine processing and release were late events inhibited by elevated levels of KCl and sucrose, by potassium channel blockers, and by proteasome inhibitors, suggesting that inflammasome formation requires a protein-degradation event and occurs downstream of LT-mediated potassium efflux. In addition, IL-18 and IL-1beta release was dependent on cell death, indicating that caspase-1-mediated cytotoxicity is independent of these cytokines. Finally, inducing NALP3-inflammasome formation in LT-resistant macrophages did not sensitize cells to LT, suggesting that general caspase-1 activation cannot account for sensitivity to LT and that a Nalp1b-mediated event is specifically required for death. Our data indicate that inflammasome formation is a contributing, but not initiating, event in LT-mediated cytotoxicity and that earlier LT-mediated events leading to ion fluxes are required for death.
Asunto(s)
Antígenos Bacterianos/fisiología , Caspasa 1/metabolismo , Macrófagos/inmunología , Complejo de la Endopetidasa Proteasomal/fisiología , Animales , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/fisiología , Toxinas Bacterianas , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Predisposición Genética a la Enfermedad , Interleucina-18/fisiología , Interleucina-1beta/fisiología , Transporte Iónico , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Ratones , Ratones Endogámicos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiologíaRESUMEN
Bacillus anthracis lethal toxin (LT) induces rapid lysis (<90 min) of murine macrophages from certain inbred strains. The mechanism for LT-induced cytolysis is currently unknown. We hypothesized that the ATP-activated macrophage P2X7 receptors implicated in nucleotide-mediated macrophage lysis could play a role in LT-mediated cytolysis and discovered that a potent P2X7 antagonist, oxidized ATP (o-ATP), protects macrophages against LT. Other P2X7 receptor antagonists, however, had no effect on LT function, while oxidized nucleotides, o-ADP, o-GTP, and o-ITP, which did not act as receptor ligands, provided protection. Cleavage of the LT substrates, the mitogen-activated protein kinases, was inhibited by o-ATP in RAW274.6 macrophages and CHO cells. We investigated the various steps in the intoxication pathway and found that binding of the protective-antigen (PA) component of LT to cells and the enzymatic proteolytic ability of the lethal factor (LF) component of LT were unaffected by o-ATP. Instead, the drug inhibited formation of the sodium dodecyl sulfate-resistant PA oligomer, which occurs in acidified endosomes, but did not prevent cell surface PA oligomerization, as evidenced by binding and translocation of LF to a protease-resistant intracellular location. We found that o-ATP also protected cells from anthrax edema toxin and diphtheria toxin, which also require an acidic environment for escape from endosomes. Confocal microscopy using pH-sensitive fluorescent dyes showed that o-ATP increased endosomal pH. Finally, BALB/cJ mice injected with o-ATP and LT were completely protected against lethality.