RESUMEN
PARP7 is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates to change their function. Here, we identify PARP7 as a negative regulator of nucleic acid sensing in tumor cells. Inhibition of PARP7 restores type I interferon (IFN) signaling responses to nucleic acids in tumor models. Restored signaling can directly inhibit cell proliferation and activate the immune system, both of which contribute to tumor regression. Oral dosing of the PARP7 small-molecule inhibitor, RBN-2397, results in complete tumor regression in a lung cancer xenograft and induces tumor-specific adaptive immune memory in an immunocompetent mouse cancer model, dependent on inducing type I IFN signaling in tumor cells. PARP7 is a therapeutic target whose inhibition induces both cancer cell-autonomous and immune stimulatory effects via enhanced IFN signaling. These data support the targeting of a monoPARP in cancer and introduce a potent and selective PARP7 inhibitor to enter clinical development.
Asunto(s)
Resistencia a Antineoplásicos/efectos de los fármacos , Interferón Tipo I/metabolismo , Neoplasias/tratamiento farmacológico , Proteínas de Transporte de Nucleósidos/genética , Proteínas de Transporte de Nucleósidos/metabolismo , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Inmunidad Adaptativa/efectos de los fármacos , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células HEK293 , Células HeLa , Humanos , Ratones , Neoplasias/genética , Neoplasias/metabolismo , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Escape del Tumor/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PARP14 is an interferon-stimulated gene that is overexpressed in multiple tumor types, influencing pro-tumor macrophage polarization as well as suppressing the antitumor inflammation response by modulating IFN-γ and IL-4 signaling. PARP14 is a 203â kDa protein that possesses a catalytic domain responsible for the transfer of mono-ADP-ribose to its substrates. PARP14 also contains three macrodomains and a WWE domain which are binding modules for mono-ADP-ribose and poly-ADP-ribose, respectively, in addition to two RNA recognition motifs. Catalytic inhibitors of PARP14 have been shown to reverse IL-4 driven pro-tumor gene expression in macrophages, however it is not clear what roles the non-enzymatic biomolecular recognition motifs play in PARP14-driven immunology and inflammation. To further understand this, we have discovered a heterobifunctional small molecule designed based on a catalytic inhibitor of PARP14 that binds in the enzyme's NAD+ -binding site and recruits cereblon to ubiquitinate it and selectively target it for degradation.
Asunto(s)
Poli(ADP-Ribosa) Polimerasas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
PARP14 has been implicated by genetic knockout studies to promote protumor macrophage polarization and suppress the antitumor inflammatory response due to its role in modulating interleukin-4 (IL-4) and interferon-γ signaling pathways. Here, we describe structure-based design efforts leading to the discovery of a potent and highly selective PARP14 chemical probe. RBN012759 inhibits PARP14 with a biochemical half-maximal inhibitory concentration of 0.003 µM, exhibits >300-fold selectivity over all PARP family members, and its profile enables further study of PARP14 biology and disease association both in vitro and in vivo. Inhibition of PARP14 with RBN012759 reverses IL-4-driven protumor gene expression in macrophages and induces an inflammatory mRNA signature similar to that induced by immune checkpoint inhibitor therapy in primary human tumor explants. These data support an immune suppressive role of PARP14 in tumors and suggest potential utility of PARP14 inhibitors in the treatment of cancer.
Asunto(s)
Antineoplásicos/farmacología , Inflamación/tratamiento farmacológico , Interleucina-4/antagonistas & inhibidores , Neoplasias Renales/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Relación Dosis-Respuesta a Droga , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Células HEK293 , Humanos , Inflamación/genética , Inflamación/metabolismo , Interleucina-4/genética , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Estructura Molecular , Poli(ADP-Ribosa) Polimerasas/genética , Células RAW 264.7 , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Poly(ADP-ribose) polymerase (PARP) enzymes use nicotinamide adenine dinucleotide (NAD+) to modify up to seven different amino acids with a single mono(ADP-ribose) unit (MARylation deposited by PARP monoenzymes) or branched poly(ADP-ribose) polymers (PARylation deposited by PARP polyenzymes). To enable the development of tool compounds for PARP monoenzymes and polyenzymes, we have developed active site probes for use in in vitro and cellular biophysical assays to characterize active site-directed inhibitors that compete for NAD+ binding. These assays are agnostic of the protein substrate for each PARP, overcoming a general lack of knowledge around the substrates for these enzymes. The in vitro assays use less enzyme than previously described activity assays, enabling discrimination of inhibitor potencies in the single-digit nanomolar range, and the cell-based assays can differentiate compounds with sub-nanomolar potencies and measure inhibitor residence time in live cells.
Asunto(s)
Colorantes Fluorescentes/química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/metabolismo , Unión Competitiva , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , NAD/química , NAD/metabolismo , Nanopartículas/química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/síntesis química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/genética , Unión Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
Poly-ADP-ribose polymerases (PARPs) are a family of enzymes responsible for transferring individual or chains of ADP-ribose subunits to substrate targets as a type of post-translational modification. PARPs regulate a wide variety of important cellular processes, ranging from DNA damage repair to antiviral response. However, most research to date has focused primarily on the polyPARPs, which catalyze the formation of ADP-ribose polymer chains, while the monoPARPs, which transfer individual ADP-ribose monomers, have not been studied as thoroughly. This is partially due to the lack of robust assays to measure mono-ADP-ribosylation in the cell. In this study, the recently developed MAR/PAR antibody has been shown to detect mono-ADP-ribosylation in cells, enabling the field to investigate the function and therapeutic potential of monoPARPs. In this study, the antibody was used in conjunction with engineered cell lines that overexpress various PARPs to establish a panel of assays to evaluate the potencies of literature-reported PARP inhibitors. These assays should be generally applicable to other PARP family members for future compound screening efforts. A convenient and generalizable workflow to identify and validate PARP substrates has been established. As an initial demonstration, aryl hydrocarbon receptor was verified as a direct PARP7 substrate and other novel substrates for this enzyme were also identified and validated. This workflow takes advantage of commercially available detection reagents and conventional mass spectrometry instrumentation and methods. Ultimately, these assays and methods will help drive research in the PARP field and benefit future therapeutics development.
Asunto(s)
ADP-Ribosilación/fisiología , Descubrimiento de Drogas/métodos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , ADP-Ribosilación/efectos de los fármacos , Descubrimiento de Drogas/tendencias , Células HeLa , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/químicaRESUMEN
In the version of this article initially published, the label (CASP4-C285A-HA) above the second and fifth lanes in the right blot in Fig. 1e is incorrect; the correct label is CASP4-C258A-HA. Also, the two labels at right above the plot in Fig. 6c were switched; the far right label should be 'Co-housed Serpinb1a-/-' (in red font) and the label just to its left (above the fourth column) should be 'Co-housed WT' (in black font). Finally, the bottom two symbols in the key to Fig. 7d were switched; the red circle should identify 1CARD-SUMO (TEV) and the blue triangle should identify 1CARD-SUMO + SERPINB1 (TEV). The errors have been corrected in the HTML and PDF versions of the article.
RESUMEN
Inflammasomes are large protein complexes that trigger host defense in cells by activating inflammatory caspases for cytokine maturation and pyroptosis. NLRP6 is a sensor protein in the nucleotide-binding domain (NBD) and leucine-rich repeat (LRR)-containing (NLR) inflammasome family that has been shown to play multiple roles in regulating inflammation and host defenses. Despite the significance of the NLRP6 inflammasome, little is known about the molecular mechanism behind its assembly and activation. Here we present cryo-EM and crystal structures of NLRP6 pyrin domain (PYD). We show that NLRP6 PYD alone is able to self-assemble into filamentous structures accompanied by large conformational changes and can recruit the ASC adaptor using PYD-PYD interactions. Using molecular dynamics simulations, we identify the surface that the NLRP6 PYD filament uses to recruit ASC PYD. We further find that full-length NLRP6 assembles in a concentration-dependent manner into wider filaments with a PYD core surrounded by the NBD and the LRR domain. These findings provide a structural understanding of inflammasome assembly by NLRP6 and other members of the NLR family.
Asunto(s)
Inflamasomas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Secuencia de Aminoácidos , Microscopía por Crioelectrón , Cristalografía por Rayos X , Humanos , Inmunidad Innata , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación Proteica , Multimerización de ProteínaRESUMEN
Inflammatory caspases (caspase-1, caspase-4, caspase-5 and caspase-11 (caspase-1/-4/-5/-11)) mediate host defense against microbial infections, processing pro-inflammatory cytokines and triggering pyroptosis. However, precise checkpoints are required to prevent their unsolicited activation. Here we report that serpin family B member 1 (SERPINB1) limited the activity of those caspases by suppressing their caspase-recruitment domain (CARD) oligomerization and enzymatic activation. While the reactive center loop of SERPINB1 inhibits neutrophil serine proteases, its carboxy-terminal CARD-binding motif restrained the activation of pro-caspase-1/-4/-5/-11. Consequently, knockdown or deletion of SERPINB1 prompted spontaneous activation of caspase-1/-4/-5/-11, release of the cytokine IL-1ß and pyroptosis, inducing elevated inflammation after non-hygienic co-housing with pet-store mice and enhanced sensitivity to lipopolysaccharide- or Acinetobacter baumannii-induced endotoxemia. Our results reveal that SERPINB1 acts as a vital gatekeeper of inflammation by restraining neutrophil serine proteases and inflammatory caspases in a genetically and functionally separable manner.
Asunto(s)
Caspasas/inmunología , Mediadores de Inflamación/inmunología , Inflamación/inmunología , Serpinas/inmunología , Animales , Caspasas/genética , Caspasas/metabolismo , Línea Celular , Células Cultivadas , Activación Enzimática/inmunología , Células HEK293 , Humanos , Inflamación/genética , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/enzimología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Piroptosis/efectos de los fármacos , Piroptosis/inmunología , Células RAW 264.7 , Interferencia de ARN , Serina Proteasas/inmunología , Serina Proteasas/metabolismo , Serpinas/genética , Serpinas/metabolismo , Células THP-1 , Células U937RESUMEN
Canonical inflammasomes are cytosolic supramolecular complexes that activate caspase-1 upon sensing extrinsic microbial invasions and intrinsic sterile stress signals. During inflammasome assembly, adaptor proteins ASC and NLRC4 recruit caspase-1 through homotypic caspase recruitment domain (CARD) interactions, leading to caspase-1 dimerization and activation. Activated caspase-1 processes proinflammatory cytokines and Gasdermin D to induce cytokine maturation and pyroptotic cell death. Here, we present cryo-electron microscopy (cryo-EM) structures of NLRC4 CARD and ASC CARD filaments mediated by conserved three types of asymmetric interactions (types I, II, and III). We find that the CARDs of these two adaptor proteins share a similar assembly pattern, which matches that of the caspase-1 CARD filament whose structure we defined previously. These data indicate a unified mechanism for downstream caspase-1 recruitment through CARD-CARD interactions by both adaptors. Using structure modeling, we further show that full-length NLRC4 assembles via two separate symmetries at its CARD and its nucleotide-binding domain (NBD), respectively.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/química , Proteínas de Unión al Calcio/química , Caspasa 1/química , Microscopía por Crioelectrón , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructura , Proteínas Adaptadoras de Señalización CARD/genética , Proteínas Adaptadoras de Señalización CARD/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Caspasa 1/genética , Caspasa 1/metabolismo , Activación Enzimática , Humanos , Inflamasomas , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismoRESUMEN
Based on the recent near-atomic structures of the PYRIN domain of ASC in the protein filament of inflammasomes and the observation that the active form of vitamin B6 (pyridoxal phosphate, P5P) modulates the self-assembly of ASC, we rationally designed an N-terminal capped nonapeptide (Nap-FFKKFKLKL, 1) to form supramolecular nanofibers consisting of α-helix. The addition of P5P to the solution of 1 results in a hydrogel almost instantly (about 4â seconds). Several other endogenous small molecules (for example, pyridoxal, folinic acid, ATP, and AMP) also convert the solution of 1 into a hydrogel. As the demonstration of correlating assemblies of peptides and the relevant protein epitopes, this work illustrates a bioinspired approach to develop supramolecular structures modulated by endogenous small molecules.
Asunto(s)
Hidrogeles/química , Inflamasomas/química , Secuencia de Aminoácidos , Epítopos/química , Microscopía Electrónica de Transmisión , Nanofibras/química , Oligopéptidos/química , Conformación ProteicaRESUMEN
Caspase-8 activation can be triggered by death receptor-mediated formation of the death-inducing signaling complex (DISC) and by the inflammasome adaptor ASC. Caspase-8 assembles with FADD at the DISC and with ASC at the inflammasome through its tandem death effector domain (tDED), which is regulated by the tDED-containing cellular inhibitor cFLIP and the viral inhibitor MC159. Here we present the caspase-8 tDED filament structure determined by cryoelectron microscopy. Extensive assembly interfaces not predicted by the previously proposed linear DED chain model were uncovered, and were further confirmed by structure-based mutagenesis in filament formation in vitro and Fas-induced apoptosis and ASC-mediated caspase-8 recruitment in cells. Structurally, the two DEDs in caspase-8 use quasi-equivalent contacts to enable assembly. Using the tDED filament structure as a template, structural analyses reveal the interaction surfaces between FADD and caspase-8 and the distinct mechanisms of regulation by cFLIP and MC159 through comingling and capping, respectively.
Asunto(s)
Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/química , Caspasa 8/química , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/química , Proteína de Dominio de Muerte Asociada a Fas/química , Proteínas Virales/química , Secuencia de Aminoácidos , Apoptosis/efectos de los fármacos , Sitios de Unión , Proteínas Adaptadoras de Señalización CARD , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Caspasa 8/genética , Caspasa 8/metabolismo , Microscopía por Crioelectrón , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/genética , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Dominio Efector de Muerte , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Expresión Génica , Humanos , Células Jurkat , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transfección , Proteínas Virales/genética , Proteínas Virales/metabolismo , Receptor fas/farmacologíaRESUMEN
Inflammasomes are cytosolic caspase-1-activation complexes that sense intrinsic and extrinsic danger signals, and trigger inflammatory responses and pyroptotic cell death. Homotypic interactions among Pyrin domains and caspase recruitment domains (CARDs) in inflammasome-complex components mediate oligomerization into filamentous assemblies. Several cytosolic proteins consisting of only interaction domains exert inhibitory effects on inflammasome assembly. In this study, we determined the structure of the human caspase-1 CARD domain (caspase-1(CARD)) filament by cryo-electron microscopy and investigated the biophysical properties of two caspase-1-like CARD-only proteins: human inhibitor of CARD (INCA or CARD17) and ICEBERG (CARD18). Our results reveal that INCA caps caspase-1 filaments, thereby exerting potent inhibition with low-nanomolar Ki on caspase-1(CARD) polymerization in vitro and inflammasome activation in cells. Whereas caspase-1(CARD) uses six complementary surfaces of three types for filament assembly, INCA is defective in two of the six interfaces and thus terminates the caspase-1 filament.
Asunto(s)
Caspasa 1/química , Secuencia de Aminoácidos , Dominio Catalítico , Línea Celular , Secuencia Conservada , Microscopía por Crioelectrón , Humanos , Enlace de Hidrógeno , Inflamasomas/metabolismo , Modelos Moleculares , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Polimerizacion , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de ProteínaRESUMEN
Myeloid cells assemble inflammasomes in response to infection or cell damage; cytosolic sensors activate pro-caspase-1, indirectly for the most part, via the adaptors ASC and NLRC4. This leads to secretion of proinflammatory cytokines and pyroptosis. To explore complex formation under physiological conditions, we generated an alpaca single domain antibody, VHHASC, which specifically recognizes the CARD of human ASC via its type II interface. VHHASC not only impairs ASC(CARD) interactions in vitro, but also inhibits inflammasome activation in response to NLRP3, AIM2, and NAIP triggers when expressed in living cells, highlighting a role of ASC in all three types of inflammasomes. VHHASC leaves the Pyrin domain of ASC functional and stabilizes a filamentous intermediate of inflammasome activation. Incorporation of VHHASC-EGFP into these structures allowed the visualization of endogenous ASC(PYD) filaments for the first time. These data revealed that cross-linking of ASC(PYD) filaments via ASC(CARD) mediates the assembly of ASC foci.
Asunto(s)
Proteínas del Citoesqueleto/inmunología , Inflamasomas/inmunología , Monocitos/inmunología , Anticuerpos de Cadena Única/farmacología , Células A549 , Proteínas Adaptadoras de Señalización CARD , Proteínas de Unión al ADN/inmunología , Femenino , Humanos , Masculino , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Proteína Inhibidora de la Apoptosis Neuronal/inmunología , Anticuerpos de Cadena Única/inmunologíaRESUMEN
Absent in melanoma 2 (AIM2) is an essential cytosolic double-stranded DNA receptor that assembles with the adaptor, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and caspase-1 to form the AIM2 inflammasome, which leads to proteolytic maturation of cytokines and pyroptotic cell death. AIM2 contains an N-terminal Pyrin domain (PYD) that interacts with ASC through PYD/PYD interactions and nucleates ASCPYD filament formation. To elucidate the molecular basis of AIM2-induced ASCPYD polymerization, we generated AIM2PYD filaments fused to green fluorescent protein (GFP) and determined its cryo-electron microscopic (cryo-EM) structure. The map showed distinct definition of helices, allowing fitting of the crystal structure. Surprisingly, the GFP-AIM2PYD filament is a 1-start helix with helical parameters distinct from those of the 3-start ASCPYD filament. However, despite the apparent symmetry difference, helical net and detailed interface analyses reveal minimal changes in subunit packing. GFP-AIM2PYD nucleated ASCPYD filament formation in comparable efficiency as untagged AIM2PYD, suggesting assembly plasticity in both AIM2PYD and ASCPYD. The DNA-binding domain of AIM2 is able to form AIM2/DNA filaments, within which the AIM2PYD is brought into proximity to template ASCPYD filament assembly. Because ASC is able to interact with many PYD-containing receptors for the formation of inflammasomes, the observed structural plasticity may be critically important for this versatility in the PYD/PYD interactions.
RESUMEN
The NLR family apoptosis inhibitory proteins (NAIPs) bind conserved bacterial ligands, such as the bacterial rod protein PrgJ, and recruit NLR family CARD-containing protein 4 (NLRC4) as the inflammasome adapter to activate innate immunity. We found that the PrgJ-NAIP2-NLRC4 inflammasome is assembled into multisubunit disk-like structures through a unidirectional adenosine triphosphatase polymerization, primed with a single PrgJ-activated NAIP2 per disk. Cryo-electron microscopy (cryo-EM) reconstruction at subnanometer resolution revealed a ~90° hinge rotation accompanying NLRC4 activation. Unlike in the related heptameric Apaf-1 apoptosome, in which each subunit needs to be conformationally activated by its ligand before assembly, a single PrgJ-activated NAIP2 initiates NLRC4 polymerization in a domino-like reaction to promote the disk assembly. These insights reveal the mechanism of signal amplification in NAIP-NLRC4 inflammasomes.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/química , Proteínas Bacterianas/química , Proteínas de Unión al Calcio/química , Inmunidad Innata , Inflamasomas/inmunología , Proteína Inhibidora de la Apoptosis Neuronal/química , Secuencia de Aminoácidos , Animales , Apoptosis , Proteínas Adaptadoras de Señalización CARD/química , Caspasa 1/química , Microscopía por Crioelectrón , Ratones , Datos de Secuencia Molecular , Polimerizacion , Mapas de Interacción de Proteínas , Estructura Terciaria de ProteínaRESUMEN
Inflammasomes mediate inflammatory and cell death responses to pathogens and cellular stress signals via activation of procaspases-1 and -8. During inflammasome assembly, activated receptors of the NLR or PYHIN family recruit the adaptor protein ASC and initiate polymerization of its pyrin domain (PYD) into filaments. We show that ASC filaments in turn nucleate procaspase-8 death effector domain (DED) filaments in vitro and in vivo. Interaction between ASC PYD and procaspase-8 tandem DEDs optimally required both DEDs and represents an unusual heterotypic interaction between domains of the death fold superfamily. Analysis of ASC PYD mutants showed that interaction surfaces that mediate procaspase-8 interaction overlap with those required for ASC self-association and interaction with the PYDs of inflammasome initiators. Our data indicate that multiple types of death fold domain filaments form at inflammasomes and that PYD/DED and homotypic PYD interaction modes are similar. Interestingly, we observed condensation of procaspase-8 filaments containing the catalytic domain, suggesting that procaspase-8 interactions within and/or between filaments may be involved in caspase-8 activation. Procaspase-8 filaments may also be relevant to apoptosis induced by death receptors.
Asunto(s)
Caspasa 8/metabolismo , Proteínas del Citoesqueleto/metabolismo , Inflamasomas/metabolismo , Apoptosis , Proteínas Adaptadoras de Señalización CARD , Caspasa 1/metabolismo , Dominio Catalítico , Muerte Celular , Células HEK293 , Humanos , Inflamación , Microscopía Fluorescente , Mutación , Unión Proteica , Transducción de SeñalRESUMEN
Inflammasomes are supramolecular signaling complexes that activate a subset of caspases known as the inflammatory caspases, an example of which is caspase 1. Upon stimulation by microbial and damage-associated signals, inflammasomes assemble to elicit the first line of host defense via the proteolytic maturation of cytokines interleukin-1ß and interleukin-18, and by induction of pyroptotic cell death. Inflammasome assembly requires activation of an upstream sensor, a downstream effector and, in most cases, an adaptor molecule such as apoptosis-associate speck-like protein containing a caspase recruitment domain (ASC). Depending on whether ASC is required, inflammasomes can be categorized into ASC-dependent and ASC-independent inflammasomes. Here, we review current understandings of the structures of inflammasomes, as probed using traditional structural methods, as well as biochemical, biophysical and single-molecule methods. The key structural scaffold for inflammasome assembly is composed of filaments of Pyrin domains and caspase recruitment domains (CARD) in the sensor, adaptor and effector components. Nucleated polymerization appears to govern the ordered assembly process from activation of a Pyrin domain-containing sensor such as AIM2 by dsDNA or NLRP3 by extracellular particulates, to recruitment of the Pyrin domain and CARD-containing adaptor ASC, and finally to activation of CARD-containing caspase 1. The underlying filamentous architecture of inflammasomes and the cooperativity in the assembly may explain the 'all-or-none' response in inflammasome activation. Inflammasomes are tightly regulated by a number of cytosolic inhibitors, which may change the morphology and assembly kinetics of inflammasomes. Biochemical and cellular studies suggest that Pyrin domain and CARD filaments possess prion-like properties in propagating inflammasome activation within and between cells.
Asunto(s)
Inflamasomas/química , Inflamasomas/metabolismo , Animales , Humanos , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Modelos Biológicos , Estructura Terciaria de ProteínaRESUMEN
Inflammasomes elicit host defense inside cells by activating caspase-1 for cytokine maturation and cell death. AIM2 and NLRP3 are representative sensor proteins in two major families of inflammasomes. The adaptor protein ASC bridges the sensor proteins and caspase-1 to form ternary inflammasome complexes, achieved through pyrin domain (PYD) interactions between sensors and ASC and through caspase activation and recruitment domain (CARD) interactions between ASC and caspase-1. We found that PYD and CARD both form filaments. Activated AIM2 and NLRP3 nucleate PYD filaments of ASC, which, in turn, cluster the CARD of ASC. ASC thus nucleates CARD filaments of caspase-1, leading to proximity-induced activation. Endogenous NLRP3 inflammasome is also filamentous. The cryoelectron microscopy structure of ASC(PYD) filament at near-atomic resolution provides a template for homo- and hetero-PYD/PYD associations, as confirmed by structure-guided mutagenesis. We propose that ASC-dependent inflammasomes in both families share a unified assembly mechanism that involves two successive steps of nucleation-induced polymerization. PAPERFLICK:
Asunto(s)
Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Inflamasomas/química , Secuencia de Aminoácidos , Proteínas Adaptadoras de Señalización CARD , Proteínas Portadoras/metabolismo , Microscopía por Crioelectrón , Proteínas de Unión al ADN , Humanos , Inflamasomas/metabolismo , Inflamasomas/ultraestructura , Interleucina-1beta/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Proteína con Dominio Pirina 3 de la Familia NLR , Proteínas Nucleares/metabolismo , Polimerizacion , Estructura Terciaria de ProteínaRESUMEN
The discovery and development of heat shock protein 70 (Hsp70) inhibitors is currently a hot topic in cancer. In the preceding paper in this issue ( 10.1021/jm401551n ), we have described structure-activity relationship studies in the first Hsp70 inhibitor class rationally designed to bind to a novel allosteric pocket located in the N-terminal domain of the protein. These ligands contained an acrylamide to take advantage of an active cysteine embedded in the allosteric pocket and acted as covalent protein modifiers upon binding. Here, we perform chemical modifications around the irreversible inhibitor scaffold to demonstrate that covalent modification is not a requirement for activity within this class of compounds. The study identifies derivative 27c, which mimics the biological effects of the irreversible inhibitors at comparable concentrations. Collectively, the back-to-back manuscripts describe the first pharmacophores that favorably and selectively interact with a never explored pocket in Hsp70 and provide a novel blueprint for a cancer-oriented development of Hsp70-directed ligands.
Asunto(s)
Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Pirimidinas/farmacología , Sitio Alostérico , Ligandos , Pirimidinas/química , Relación Estructura-ActividadRESUMEN
Absent in melanoma 2 (AIM2) is a cytoplasmic double-stranded DNA sensor involved in innate immunity. It uses its C-terminal HIN domain for recognizing double-stranded DNA and its N-terminal pyrin domain (PYD) for eliciting downstream effects through recruitment and activation of apoptosis-associated Speck-like protein containing CARD (ASC). ASC in turn recruits caspase-1 and/or caspase-11 to form the AIM2 inflammasome. The activated caspases process proinflammatory cytokines IL-1ß and IL-18 and induce the inflammatory form of cell death pyroptosis. Here we show that AIM PYD (AIM2(PYD)) self-oligomerizes. We notice significant sequence homology of AIM2(PYD) with the hydrophobic patches of death effector domain (DED)-containing proteins and confirm that mutations on these residues disrupt AIM2(PYD) self-association. The crystal structure at 1.82Å resolution of such a mutant, F27G of AIM2(PYD), shows the canonical six-helix (H1-H6) bundle fold in the death domain superfamily. In contrast to the wild-type AIM2(PYD) structure crystallized in fusion with the large maltose-binding protein tag, the H2-H3 region of the AIM2(PYD) F27G is well defined with low B-factors. Structural analysis shows that the conserved hydrophobic patches engage in a type I interaction that has been observed in DED/DED and other death domain superfamily interactions. While previous mutagenesis studies of PYDs point to the involvement of charged interactions, our results reveal the importance of hydrophobic interactions in the same interfaces. These centrally localized hydrophobic residues within fairly charged patches may form the hot spots in AIM2(PYD) self-association and may represent a common mode of PYD/PYD interactions in general.