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1.
Cell Chem Biol ; 2024 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-38991619

RESUMEN

Mounting evidence indicates that proteotoxic stress is a primary activator of the CARD8 inflammasome, but the complete array of signals that control this inflammasome have not yet been established. Notably, we recently discovered that several hydrophobic radical-trapping antioxidants (RTAs), including JSH-23, potentiate CARD8 inflammasome activation through an unknown mechanism. Here, we report that these RTAs directly alkylate several cysteine residues in the N-terminal disordered region of CARD8. These hydrophobic modifications destabilize the repressive CARD8 N-terminal fragment and accelerate its proteasome-mediated degradation, thereby releasing the inflammatory CARD8 C-terminal fragment from autoinhibition. Consistently, we also found that unrelated (non-RTA) hydrophobic electrophiles as well as genetic mutation of the CARD8 cysteine residues to isoleucines similarly potentiate inflammasome activation. Overall, our results not only provide further evidence that protein folding stress is a key CARD8 inflammasome-activating signal, but also indicate that the N-terminal cysteines can play key roles in tuning the response to this stress.

2.
Cell Chem Biol ; 31(5): 955-961.e4, 2024 May 16.
Artículo en Inglés | MEDLINE | ID: mdl-38215746

RESUMEN

NLRP1 is an innate immune receptor that detects pathogen-associated signals, assembles into a multiprotein structure called an inflammasome, and triggers a proinflammatory form of cell death called pyroptosis. We previously discovered that the oxidized, but not the reduced, form of thioredoxin-1 directly binds to NLRP1 and represses inflammasome formation. However, the molecular basis for NLRP1's selective association with only the oxidized form of TRX1 has not yet been established. Here, we leveraged AlphaFold-Multimer, site-directed mutagenesis, thiol-trapping experiments, and mass spectrometry to reveal that a specific cysteine residue (C427 in humans) on NLRP1 forms a transient disulfide bond with oxidized TRX1. Overall, this work demonstrates how NLRP1 monitors the cellular redox state, further illuminating an unexpected connection between the intracellular redox potential and the innate immune system.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Disulfuros , Proteínas NLR , Oxidación-Reducción , Tiorredoxinas , Humanos , Disulfuros/química , Disulfuros/metabolismo , Tiorredoxinas/metabolismo , Tiorredoxinas/química , Proteínas NLR/metabolismo , Proteínas NLR/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Células HEK293 , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Reguladoras de la Apoptosis/química , Inflamasomas/metabolismo , Cisteína/metabolismo , Cisteína/química
3.
bioRxiv ; 2023 Sep 28.
Artículo en Inglés | MEDLINE | ID: mdl-37808697

RESUMEN

NLRP1 is an innate immune receptor that detects pathogen-associated signals, assembles into a multiprotein structure called an inflammasome, and triggers a proinflammatory form of cell death called pyroptosis. We previously discovered that the oxidized, but not the reduced, form of thioredoxin-1 directly binds to NLRP1 and represses inflammasome formation. However, the molecular basis for NLRP1's selective association with only the oxidized form of TRX1 has not yet been established. Here, we leveraged Alphafold-Multimer, site-directed mutagenesis, thiol-trapping experiments, and mass spectrometry to reveal that a specific cysteine residue (C427 in humans) on NLRP1 forms a transient disulfide bond with oxidized TRX1. Overall, this work demonstrates how NLRP1 monitors the cellular redox state, further illuminating an unexpected connection between the intracellular redox potential and the innate immune system.

4.
J Med Chem ; 66(4): 2589-2607, 2023 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-36724486

RESUMEN

Inflammasomes are innate immune signaling platforms that trigger pyroptotic cell death. NLRP1 and CARD8 are related human inflammasomes that detect similar danger signals, but NLRP1 has a higher activation threshold and triggers a more inflammatory form of pyroptosis. Both sense the accumulation of intracellular peptides with Xaa-Pro N-termini, but Xaa-Pro peptides on their own without a second danger signal only activate the CARD8 inflammasome. We recently reported that a dual inhibitor of the Xaa-Pro-cleaving M24B aminopeptidases PEPD and XPNPEP1 called CQ31 selectively activates the CARD8 inflammasome by inducing the build-up of Xaa-Pro peptides. Here, we performed structure-activity relationship studies on CQ31 to develop the optimized dual PEPD/XPNPEP1 inhibitor CQ80 that more effectively induces CARD8 inflammasome activation. We anticipate that CQ80 will become a valuable tool to study the basic biology and therapeutic potential of selective CARD8 inflammasome activation.


Asunto(s)
Aminopeptidasas , Inflamasomas , Humanos , Inflamasomas/metabolismo , Aminopeptidasas/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Transducción de Señal , Piroptosis , Proteínas de Neoplasias/metabolismo , Proteínas Adaptadoras de Señalización CARD/metabolismo
5.
Nat Chem Biol ; 19(2): 159-167, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36253549

RESUMEN

The human gut bacterial genotoxin colibactin is a possible key driver of colorectal cancer (CRC) development. Understanding colibactin's biological effects remains difficult owing to the instability of the proposed active species and the complexity of the gut microbiota. Here, we report small molecule boronic acid inhibitors of colibactin biosynthesis. Designed to mimic the biosynthetic precursor precolibactin, these compounds potently inhibit the colibactin-activating peptidase ClbP. Using biochemical assays and crystallography, we show that they engage the ClbP binding pocket, forming a covalent bond with the catalytic serine. These inhibitors reproduce the phenotypes observed in a clbP deletion mutant and block the genotoxic effects of colibactin on eukaryotic cells. The availability of ClbP inhibitors will allow precise, temporal control over colibactin production, enabling further study of its contributions to CRC. Finally, application of our inhibitors to related peptidase-encoding pathways highlights the power of chemical tools to probe natural product biosynthesis.


Asunto(s)
Microbioma Gastrointestinal , Policétidos , Humanos , Mutágenos/metabolismo , Mutágenos/toxicidad , Escherichia coli/metabolismo , Policétidos/química , Péptido Hidrolasas/química
6.
J Biol Chem ; 298(7): 102032, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35580636

RESUMEN

CARD8 is a pattern-recognition receptor that forms a caspase-1-activating inflammasome. CARD8 undergoes constitutive autoproteolysis, generating an N-terminal (NT) fragment with a disordered region and a ZU5 domain and a C-terminal (CT) fragment with UPA and CARD domains. Dipeptidyl peptidase 8 and dipeptidyl peptidase 9 inhibitors, including Val-boroPro, accelerate the degradation of the NT fragment via a poorly characterized proteasome-mediated pathway, thereby releasing the inflammatory CT fragment from autoinhibition. Here, we show that the core 20S proteasome, which degrades disordered and misfolded proteins independent of ubiquitin modification, controls activation of the CARD8 inflammasome. In unstressed cells, we discovered that the 20S proteasome degrades just the NT disordered region, leaving behind the folded ZU5, UPA, and CARD domains to act as an inhibitor of inflammasome assembly. However, in Val-boroPro-stressed cells, we show the 20S proteasome degrades the entire NT fragment, perhaps due to ZU5 domain unfolding, freeing the CT fragment from autoinhibition. Taken together, these results show that the susceptibility of the CARD8 NT domain to 20S proteasome-mediated degradation controls inflammasome activation.


Asunto(s)
Proteínas Adaptadoras de Señalización CARD , Inflamasomas , Complejo de la Endopetidasa Proteasomal , Proteínas Adaptadoras de Señalización CARD/metabolismo , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/antagonistas & inhibidores , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Humanos , Inflamasomas/metabolismo , Proteínas de Neoplasias/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitinas/metabolismo
7.
Invest Ophthalmol Vis Sci ; 63(3): 2, 2022 Mar 02.
Artículo en Inglés | MEDLINE | ID: mdl-35238869

RESUMEN

PURPOSE: Inflammasomes are multiprotein complexes that detect danger-associated signals and trigger an immunostimulatory form of cell death called pyroptosis. NLRP1 is an innate immune receptor that assembles into an inflammasome, but the primary cell types in which NLRP1 is functional have not yet been fully established. Mutations in NLRP1 are associated with diseases of barrier epithelial tissues, including skin lesions and corneal intraepithelial dyskeratosis, suggesting that NLRP1 functions within the eye. Here, we investigated the expression and activity of the NLRP1 inflammasome in primary human corneal epithelial (pHCE) cells. METHODS: The small molecule Val-boroPro (VbP) activates the NLRP1 inflammasome. Proteasome (bortezomib, MG132) and caspase-1 (VX-765, Z-VAD-FMK) inhibitors block NLRP1 activation and downstream pyroptosis, respectively. Here, we treated pHCE cells with VbP alone or in combination proteasome inhibitors and caspase-1 inhibitors. We assessed NLRP1 expression and hallmarks of pyroptosis, including lytic cell rupture, cytokine processing and release, and gasdermin D (GSDMD) processing. RESULTS: VbP triggered pyroptosis in pHCE cells, as determined by cytokine secretion, GSDMD processing, and lactate dehydrogenase (LDH) release. Proteasome and caspase-1 inhibitors completely blocked this pyroptotic cell death. In contrast, other primary ocular epithelial cells did not undergo NLRP1-dependent pyroptosis. CONCLUSIONS: Our findings demonstrate that NLRP1 forms a functional inflammasome in pHCE cells. Importantly, these data reveal that NLRP1 is a key innate immune sensor of the corneal epithelium, and moreover indicate how aberrant inflammasome activation causes corneal damage. Blockade of NLRP1 signaling may benefit patients with hyperactive NLRP1 mutations and warrants further investigation.


Asunto(s)
Células Epiteliales , Inflamasomas , Proteínas NLR , Piroptosis , Caspasa 1/metabolismo , Córnea/citología , Citocinas , Células Epiteliales/metabolismo , Humanos , Inflamasomas/metabolismo , Complejo de la Endopetidasa Proteasomal
8.
Nat Chem Biol ; 18(5): 565-574, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35165443

RESUMEN

Inflammasomes are multiprotein complexes that sense intracellular danger signals and induce pyroptosis. CARD8 and NLRP1 are related inflammasomes that are repressed by the enzymatic activities and protein structures of the dipeptidyl peptidases 8 and 9 (DPP8/9). Potent DPP8/9 inhibitors such as Val-boroPro (VbP) activate both NLRP1 and CARD8, but chemical probes that selectively activate only one have not been identified. Here we report a small molecule called CQ31 that selectively activates CARD8. CQ31 inhibits the M24B aminopeptidases prolidase (PEPD) and Xaa-Pro aminopeptidase 1 (XPNPEP1), leading to the accumulation of proline-containing peptides that inhibit DPP8/9 and thereby activate CARD8. NLRP1 is distinct from CARD8 in that it directly contacts DPP8/9's active site; these proline-containing peptides, unlike VbP, do not disrupt this repressive interaction and thus do not activate NLRP1. We expect that CQ31 will now become a valuable tool to study CARD8 biology.


Asunto(s)
Proteínas Adaptadoras de Señalización CARD , Inflamasomas , Aminopeptidasas/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Adaptadoras de Señalización CARD/metabolismo , Proteínas de Neoplasias , Prolina
9.
Immunity ; 54(7): 1392-1404.e10, 2021 07 13.
Artículo en Inglés | MEDLINE | ID: mdl-34019797

RESUMEN

CARD8 detects intracellular danger signals and forms a caspase-1 activating inflammasome. Like the related inflammasome sensor NLRP1, CARD8 autoprocesses into noncovalently associated N-terminal (NT) and C-terminal (CT) fragments and binds the cellular dipeptidyl peptidases DPP8 and 9 (DPP8/9). Certain danger-associated signals, including the DPP8/9 inhibitor Val-boroPro (VbP) and HIV protease, induce proteasome-mediated NT degradation and thereby liberate the inflammasome-forming CT. Here, we report cryoelectron microscopy (cryo-EM) structures of CARD8 bound to DPP9, revealing a repressive ternary complex consisting of DPP9, full-length CARD8, and CARD8-CT. Unlike NLRP1-CT, CARD8-CT does not interact with the DPP8/9 active site and is not directly displaced by VbP. However, larger DPP8/9 active-site probes can directly weaken this complex in vitro, and VbP itself nevertheless appears to disrupt this complex, perhaps indirectly, in cells. Thus, DPP8/9 inhibitors can activate the CARD8 inflammasome by promoting CARD8 NT degradation and by weakening ternary complex stability.


Asunto(s)
Proteínas Adaptadoras de Señalización CARD/metabolismo , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Inflamasomas/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Caspasa 1/metabolismo , Dominio Catalítico/fisiología , Línea Celular , Microscopía por Crioelectrón/métodos , Células HEK293 , Humanos , Proteolisis , Células Sf9
10.
Mol Cell ; 81(3): 423-425, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33545058

RESUMEN

Recent studies provide evidence that two chemically and mechanistically distinct signals activate the human NLRP1 inflammasome, challenging the concept that it-like other mammalian inflammasomes characterized thus far-evolved to detect and respond to a single danger-associated molecular pattern.


Asunto(s)
Inflamasomas , Péptido Hidrolasas , Proteasas Virales 3C , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Cisteína Endopeptidasas , Humanos , Inflamasomas/metabolismo , Proteínas NLR , Proteínas Virales
11.
Nat Commun ; 12(1): 189, 2021 01 08.
Artículo en Inglés | MEDLINE | ID: mdl-33420033

RESUMEN

NLRP1 and CARD8 are related cytosolic sensors that upon activation form supramolecular signalling complexes known as canonical inflammasomes, resulting in caspase-1 activation, cytokine maturation and/or pyroptotic cell death. NLRP1 and CARD8 use their C-terminal (CT) fragments containing a caspase recruitment domain (CARD) and the UPA (conserved in UNC5, PIDD, and ankyrins) subdomain for self-oligomerization, which in turn form the platform to recruit the inflammasome adaptor ASC (apoptosis-associated speck-like protein containing a CARD) or caspase-1, respectively. Here, we report cryo-EM structures of NLRP1-CT and CARD8-CT assemblies, in which the respective CARDs form central helical filaments that are promoted by oligomerized, but flexibly linked, UPAs surrounding the filaments. Through biochemical and cellular approaches, we demonstrate that the UPA itself reduces the threshold needed for NLRP1-CT and CARD8-CT filament formation and signalling. Structural analyses provide insights on the mode of ASC recruitment by NLRP1-CT and the contrasting direct recruitment of caspase-1 by CARD8-CT. We also discover that subunits in the central NLRP1CARD filament dimerize with additional exterior CARDs, which roughly doubles its thickness and is unique among all known CARD filaments. Finally, we engineer and determine the structure of an ASCCARD-caspase-1CARD octamer, which suggests that ASC uses opposing surfaces for NLRP1, versus caspase-1, recruitment. Together these structures capture the architecture and specificity of the active NLRP1 and CARD8 inflammasomes in addition to key heteromeric CARD-CARD interactions governing inflammasome signalling.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Ancirinas/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Adaptadoras de Señalización CARD/metabolismo , Inflamasomas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Ancirinas/química , Apoptosis , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Adaptadoras de Señalización CARD/química , Proteínas Adaptadoras de Señalización CARD/genética , Caspasa 1/metabolismo , Dominio de Reclutamiento y Activación de Caspasas , Microscopía por Crioelectrón , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/química , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Células HEK293 , Humanos , Inflamasomas/química , Inflamasomas/ultraestructura , Modelos Moleculares , Proteínas NLR , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal
12.
Cell Rep ; 33(2): 108264, 2020 10 13.
Artículo en Inglés | MEDLINE | ID: mdl-33053349

RESUMEN

Several cytosolic pattern-recognition receptors (PRRs) form multiprotein complexes called canonical inflammasomes in response to intracellular danger signals. Canonical inflammasomes recruit and activate caspase-1 (CASP1), which in turn cleaves and activates inflammatory cytokines and gasdermin D (GSDMD), inducing pyroptotic cell death. Inhibitors of the dipeptidyl peptidases DPP8 and DPP9 (DPP8/9) activate both the human NLRP1 and CARD8 inflammasomes. NLRP1 and CARD8 have different N-terminal regions but have similar C-terminal regions that undergo autoproteolysis to generate two non-covalently associated fragments. Here, we show that DPP8/9 inhibition activates a proteasomal degradation pathway that targets disordered and misfolded proteins for destruction. CARD8's N terminus contains a disordered region of ∼160 amino acids that is recognized and destroyed by this degradation pathway, thereby freeing its C-terminal fragment to activate CASP1 and induce pyroptosis. Thus, CARD8 serves as an alarm to signal the activation of a degradation pathway for disordered and misfolded proteins.


Asunto(s)
Proteínas Adaptadoras de Señalización CARD/química , Proteínas Adaptadoras de Señalización CARD/metabolismo , Inflamasomas/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Animales , Ácidos Borónicos/farmacología , Dipéptidos/farmacología , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Células HEK293 , Humanos , Lisina/metabolismo , Ratones , Proteolisis , Proteostasis , Células RAW 264.7 , Células THP-1
13.
Cell Death Dis ; 11(8): 628, 2020 08 14.
Artículo en Inglés | MEDLINE | ID: mdl-32796818

RESUMEN

Canonical inflammasomes are innate immune signaling platforms that are formed in response to intracellular pathogen-associated signals and trigger caspase-1-dependent pyroptosis. Inflammasome formation and signaling is thought to mainly occur in myeloid cells, and in particular monocytes and macrophages. Here we show that small molecule inhibitors of dipeptidyl peptidases 8 and 9 (DPP8/9), which activate the related CARD8 and NLRP1 inflammasomes, also activate pyroptosis in human and rodent resting lymphocytes. We found that both CD4+ and CD8+ T cells were particularly sensitive to these inhibitors, although the sensitivity of T cells, like macrophages, varied considerably between species. In human T cells, we show that CARD8 mediates DPP8/9 inhibitor-induced pyroptosis. Intriguingly, although activated human T cells express the key proteins known to be required for CARD8-mediated pyroptosis, these cells were completely resistant to DPP8/9 inhibitors. Overall, these data show that resting lymphoid cells can activate at least one inflammasome, revealing additional cell types and states poised to undergo rapid pyroptotic cell death in response to danger-associated signals.


Asunto(s)
Proteínas Adaptadoras de Señalización CARD/metabolismo , Ciclo Celular , Dipeptidasas/antagonistas & inhibidores , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/antagonistas & inhibidores , Inflamasomas/metabolismo , Linfocitos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Ciclo Celular/efectos de los fármacos , Células Cultivadas , Dipeptidasas/metabolismo , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Humanos , Activación de Linfocitos/efectos de los fármacos , Linfocitos/efectos de los fármacos , Ratones , Proteínas NLR , Inhibidores de Proteasas/farmacología , Piroptosis/efectos de los fármacos , Ratas
14.
Immunol Rev ; 297(1): 13-25, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32558991

RESUMEN

Inflammasomes are multiprotein complexes that activate inflammatory cytokines and induce pyroptosis in response to intracellular danger-associated signals. NLRP1 and CARD8 are related germline-encoded pattern recognition receptors that form inflammasomes, but their activation mechanisms and biological purposes have not yet been fully established. Both NLRP1 and CARD8 undergo post-translational autoproteolysis to generate two non-covalently associated polypeptide chains. NLRP1 and CARD8 activators induce the proteasome-mediated destruction of the N-terminal fragment, liberating the C-terminal fragment to form an inflammasome. Here, we review the danger-associated stimuli that have been reported to activate NLRP1 and/or CARD8, including anthrax lethal toxin, Toxoplasma gondii, Shigella flexneri and the small molecule DPP8/9 inhibitor Val-boroPro, focusing on recent mechanistic insights and highlighting unresolved questions. In addition, we discuss the recently identified disease-associated mutations in NLRP1 and CARD8, the potential role that DPP9's protein structure plays in inflammasome regulation, and the emerging link between NLRP1 and metabolism. Finally, we summarize all of this latest research and consider the possible biological purposes of these enigmatic inflammasomes.


Asunto(s)
Proteínas Adaptadoras de Señalización CARD , Inflamasomas , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Adaptadoras de Señalización CARD/metabolismo , Humanos , Inflamasomas/metabolismo , Proteínas NLR , Proteínas de Neoplasias/metabolismo
15.
Bioorg Med Chem Lett ; 29(24): 126753, 2019 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-31679971

RESUMEN

The oral K+-sparing diuretic amiloride shows anti-cancer side-activities in multiple rodent models. These effects appear to arise, at least in part, through moderate inhibition of the urokinase-type plasminogen activator (uPA, Ki = 2.4 µM), a pro-metastatic trypsin-like serine protease that is upregulated in many aggressive solid malignancies. In applying the selective optimization of side-activity (SOSA) approach, a focused library of twenty two 6-substituted amiloride derivatives were prepared, with multiple examples displaying uPA inhibitory potencies in the nM range. X-ray co-crystal structures revealed that the potency increases relative to amiloride arise from increased occupancy of uPA's S1ß subsite by the appended 6-substituents. Leading compounds were shown to have high selectivity over related trypsin-like serine proteases and no diuretic or anti-kaliuretic effects in rats. Compound 15 showed anti-metastatic effects in a xenografted mouse model of late-stage lung metastasis.


Asunto(s)
Amilorida/análogos & derivados , Amilorida/uso terapéutico , Diuréticos/uso terapéutico , Metástasis de la Neoplasia/tratamiento farmacológico , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidores , Amilorida/farmacología , Diuréticos/farmacología , Humanos , Relación Estructura-Actividad
16.
ACS Chem Biol ; 14(11): 2424-2429, 2019 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-31525884

RESUMEN

Inflammasomes are multiprotein complexes formed in response to pathogens. NLRP1 and CARD8 are related proteins that form inflammasomes, but the pathogen-associated signal(s) and the molecular mechanisms controlling their activation have not been established. Inhibitors of the serine dipeptidyl peptidases DPP8 and DPP9 (DPP8/9) activate both NLRP1 and CARD8. Interestingly, DPP9 binds directly to NLRP1 and CARD8, and this interaction may contribute to the inhibition of NLRP1. Here, we use activity-based probes, reconstituted inflammasome assays, and mass spectrometry-based proteomics to further investigate the DPP9-CARD8 interaction. We show that the DPP9-CARD8 interaction, unlike the DPP9-NLRP1 interaction, is not disrupted by DPP9 inhibitors or CARD8 mutations that block autoproteolysis. Moreover, wild-type, but not catalytically inactive mutant, DPP9 rescues CARD8-mediated cell death in DPP9 knockout cells. Together, this work reveals that DPP9's catalytic activity and not its binding to CARD8 restrains the CARD8 inflammasome and thus suggests the binding interaction likely serves some other biological purpose.


Asunto(s)
Proteínas Adaptadoras de Señalización CARD/metabolismo , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Inflamasomas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Dipeptidasas/metabolismo , Células HEK293 , Humanos , Mutación , Proteínas NLR , Organofluorofosfonatos/metabolismo , Inhibidores de Proteasas/metabolismo , Unión Proteica , Conformación Proteica , Transducción de Señal
17.
J Med Chem ; 61(18): 8299-8320, 2018 09 27.
Artículo en Inglés | MEDLINE | ID: mdl-30130401

RESUMEN

Metastasis is the cause of death in the majority (∼90%) of malignant cancers. The oral potassium-sparing diuretic amiloride and its 5-substituted derivative 5 -N, N-(hexamethylene)amiloride (HMA) reportedly show robust antitumor/metastasis effects in multiple in vitro and animal models. These effects are likely due, at least in part, to inhibition of the urokinase plasminogen activator (uPA), a key protease determinant of cell invasiveness and metastasis. This study reports the discovery of 6-substituted HMA analogs that show nanomolar potency against uPA, high selectivity over related trypsin-like serine proteases, and minimal inhibitory effects against epithelial sodium channels (ENaC), the diuretic and antikaliuretic target of amiloride. Reductions in lung metastases were demonstrated for two analogs in a late-stage experimental mouse metastasis model, and one analog completely inhibited formation of liver metastases in an orthotopic xenograft mouse model of pancreatic cancer. The results support further evaluation of 6-substituted HMA derivatives as uPA-targeting anticancer drugs.


Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Diuresis/efectos de los fármacos , Descubrimiento de Drogas , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidores , Amilorida/química , Animales , Movimiento Celular , Proliferación Celular , Cristalografía por Rayos X , Diuréticos/química , Diuréticos/farmacología , Femenino , Humanos , Neoplasias Pulmonares/secundario , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Modelos Moleculares , Estructura Molecular , Neoplasias Pancreáticas/patología , Potasio/metabolismo , Conformación Proteica , Sodio/metabolismo , Relación Estructura-Actividad , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
18.
Nat Med ; 24(8): 1151-1156, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29967349

RESUMEN

Small-molecule inhibitors of the serine dipeptidases DPP8 and DPP9 (DPP8/9) induce a lytic form of cell death called pyroptosis in mouse and human monocytes and macrophages1,2. In mouse myeloid cells, Dpp8/9 inhibition activates the inflammasome sensor Nlrp1b, which in turn activates pro-caspase-1 to mediate cell death3, but the mechanism of DPP8/9 inhibitor-induced pyroptosis in human myeloid cells is not yet known. Here we show that the CARD-containing protein CARD8 mediates DPP8/9 inhibitor-induced pro-caspase-1-dependent pyroptosis in human myeloid cells. We further show that DPP8/9 inhibitors induce pyroptosis in the majority of human acute myeloid leukemia (AML) cell lines and primary AML samples, but not in cells from many other lineages, and that these inhibitors inhibit human AML progression in mouse models. Overall, this work identifies an activator of CARD8 in human cells and indicates that its activation by small-molecule DPP8/9 inhibitors represents a new potential therapeutic strategy for AML.


Asunto(s)
Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Inhibidores de Proteasas/uso terapéutico , Piroptosis/efectos de los fármacos , Proteínas Adaptadoras de Señalización CARD/metabolismo , Caspasa 1/metabolismo , Línea Celular Tumoral , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Progresión de la Enfermedad , Células HEK293 , Humanos , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología
19.
Cell Chem Biol ; 25(3): 262-267.e5, 2018 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-29396289

RESUMEN

Val-boroPro (PT-100, Talabostat) induces powerful anti-tumor immune responses in syngeneic cancer models, but its mechanism of action has not yet been established. Val-boroPro is a non-selective inhibitor of post-proline-cleaving serine proteases, and the inhibition of the highly related cytosolic serine proteases Dpp8 and Dpp9 (Dpp8/9) by Val-boroPro was recently demonstrated to trigger an immunostimulatory form of programmed cell death known as pyroptosis selectively in monocytes and macrophages. Here we show that Dpp8/9 inhibition activates the inflammasome sensor protein Nlrp1b, which in turn activates pro-caspase-1 to mediate pyroptosis. This work reveals a previously unrecognized mechanism for activating an innate immune pattern recognition receptor and suggests that Dpp8/9 serve as an intracellular checkpoint to restrain Nlrp1b and the innate immune system.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Dipeptidasas/metabolismo , Inflamasomas/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/química , Ácidos Borónicos/química , Ácidos Borónicos/metabolismo , Ácidos Borónicos/farmacología , Caspasa 1/metabolismo , Dipeptidasas/antagonistas & inhibidores , Dipéptidos/química , Dipéptidos/metabolismo , Dipéptidos/farmacología , Femenino , Células HEK293 , Humanos , Inmunidad Innata/efectos de los fármacos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Piroptosis/efectos de los fármacos , Células RAW 264.7
20.
Cell Chem Biol ; 24(12): 1523-1536.e4, 2017 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-29107700

RESUMEN

Rhomboid-family intramembrane proteases regulate important biological processes and have been associated with malaria, cancer, and Parkinson's disease. However, due to the lack of potent, selective, and pharmacologically compliant inhibitors, the wide therapeutic potential of rhomboids is currently untapped. Here, we bridge this gap by discovering that peptidyl α-ketoamides substituted at the ketoamide nitrogen by hydrophobic groups are potent rhomboid inhibitors active in the nanomolar range, surpassing the currently used rhomboid inhibitors by up to three orders of magnitude. Such peptidyl ketoamides show selectivity for rhomboids, leaving most human serine hydrolases unaffected. Crystal structures show that these compounds bind the active site of rhomboid covalently and in a substrate-like manner, and kinetic analysis reveals their reversible, slow-binding, non-competitive mechanism. Since ketoamides are clinically used pharmacophores, our findings uncover a straightforward modular way for the design of specific inhibitors of rhomboid proteases, which can be widely applicable in cell biology and drug discovery.


Asunto(s)
Diseño de Fármacos , Péptido Hidrolasas/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Bacterias Gramnegativas/enzimología , Bacterias Grampositivas/enzimología , Modelos Moleculares , Conformación Molecular , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/química
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