RESUMEN
The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.
Asunto(s)
Aminopiridinas/química , Aminopiridinas/farmacología , Indazoles/química , Indazoles/farmacología , Fenoles/química , Fenoles/farmacología , Piridinas/química , Piridinas/farmacología , Peptidasa Específica de Ubiquitina 7/antagonistas & inhibidores , Ubiquitina/metabolismo , Animales , Unión Competitiva , Línea Celular Tumoral , Sinergismo Farmacológico , Femenino , Humanos , Ratones , Ratones SCID , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/patología , Unión Proteica , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Especificidad por Sustrato , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina/química , Peptidasa Específica de Ubiquitina 7/química , Peptidasa Específica de Ubiquitina 7/deficiencia , Peptidasa Específica de Ubiquitina 7/metabolismoRESUMEN
The endoplasmic reticulum (ER) is the primary organelle for folding and maturation of secretory and transmembrane proteins. Inability to meet protein-folding demand leads to "ER stress," and activates IRE1α, an ER transmembrane kinase-endoribonuclease (RNase). IRE1α promotes adaptation through splicing Xbp1 mRNA or apoptosis through incompletely understood mechanisms. Here, we found that sustained IRE1α RNase activation caused rapid decay of select microRNAs (miRs -17, -34a, -96, and -125b) that normally repress translation of Caspase-2 mRNA, and thus sharply elevates protein levels of this initiator protease of the mitochondrial apoptotic pathway. In cell-free systems, recombinant IRE1α endonucleolytically cleaved microRNA precursors at sites distinct from DICER. Thus, IRE1α regulates translation of a proapoptotic protein through terminating microRNA biogenesis, and noncoding RNAs are part of the ER stress response.
Asunto(s)
Caspasa 2/genética , Caspasa 2/metabolismo , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Estrés del Retículo Endoplásmico , Endorribonucleasas/metabolismo , MicroARNs/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Regiones no Traducidas 3' , Animales , Apoptosis , Brefeldino A/farmacología , Sistema Libre de Células , Células Cultivadas , Regulación hacia Abajo , Retículo Endoplásmico/metabolismo , Endorribonucleasas/química , Endorribonucleasas/genética , Activación Enzimática , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Proteínas Mutantes , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación hacia ArribaRESUMEN
When unfolded proteins accumulate to irremediably high levels within the endoplasmic reticulum (ER), intracellular signaling pathways called the unfolded protein response (UPR) become hyperactivated to cause programmed cell death. We discovered that thioredoxin-interacting protein (TXNIP) is a critical node in this "terminal UPR." TXNIP becomes rapidly induced by IRE1α, an ER bifunctional kinase/endoribonuclease (RNase). Hyperactivated IRE1α increases TXNIP mRNA stability by reducing levels of a TXNIP destabilizing microRNA, miR-17. In turn, elevated TXNIP protein activates the NLRP3 inflammasome, causing procaspase-1 cleavage and interleukin 1ß (IL-1ß) secretion. Txnip gene deletion reduces pancreatic ß cell death during ER stress and suppresses diabetes caused by proinsulin misfolding in the Akita mouse. Finally, small molecule IRE1α RNase inhibitors suppress TXNIP production to block IL-1ß secretion. In summary, the IRE1α-TXNIP pathway is used in the terminal UPR to promote sterile inflammation and programmed cell death and may be targeted to develop effective treatments for cell degenerative diseases.
Asunto(s)
Apoptosis/fisiología , Proteínas Portadoras/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Endorribonucleasas/metabolismo , Inflamasomas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Tiorredoxinas/metabolismo , Respuesta de Proteína Desplegada/fisiología , Animales , Western Blotting , Línea Celular , Cartilla de ADN/genética , Citometría de Flujo , Humanos , Interleucina-1beta/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
During endoplasmic reticulum (ER) stress, homeostatic signaling through the unfolded protein response (UPR) augments ER protein-folding capacity. If homeostasis is not restored, the UPR triggers apoptosis. We found that the ER transmembrane kinase/endoribonuclease (RNase) IRE1alpha is a key component of this apoptotic switch. ER stress induces IRE1alpha kinase autophosphorylation, activating the RNase to splice XBP1 mRNA and produce the homeostatic transcription factor XBP1s. Under ER stress--or forced autophosphorylation--IRE1alpha's RNase also causes endonucleolytic decay of many ER-localized mRNAs, including those encoding chaperones, as early events culminating in apoptosis. Using chemical genetics, we show that kinase inhibitors bypass autophosphorylation to activate the RNase by an alternate mode that enforces XBP1 splicing and averts mRNA decay and apoptosis. Alternate RNase activation by kinase-inhibited IRE1alpha can be reconstituted in vitro. We propose that divergent cell fates during ER stress hinge on a balance between IRE1alpha RNase outputs that can be tilted with kinase inhibitors to favor survival.
Asunto(s)
Endorribonucleasas/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Células/metabolismo , Retículo Endoplásmico/metabolismo , Insulina/genética , Complejos Multienzimáticos , Pliegue de Proteína , Proteínas Serina-Treonina Quinasas , Estabilidad del ARN , Ratas , RibonucleasasRESUMEN
The accumulation of misfolded proteins stresses the endoplasmic reticulum (ER) and triggers cell death through activation of the multidomain proapoptotic BCL-2 proteins BAX and BAK at the outer mitochondrial membrane. The signaling events that connect ER stress with the mitochondrial apoptotic machinery remain unclear, despite evidence that deregulation of this pathway contributes to cell loss in many human degenerative diseases. In order to "trap" and identify the apoptotic signals upstream of mitochondrial permeabilization, we challenged Bax-/- Bak-/- mouse embryonic fibroblasts with pharmacological inducers of ER stress. We found that ER stress induces proteolytic activation of the BH3-only protein BID as a critical apoptotic switch. Moreover, we identified caspase-2 as the premitochondrial protease that cleaves BID in response to ER stress and showed that resistance to ER stress-induced apoptosis can be conferred by inhibiting caspase-2 activity. Our work defines a novel signaling pathway that couples the ER and mitochondria and establishes a principal apoptotic effector downstream of ER stress.
Asunto(s)
Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Caspasa 2/metabolismo , Retículo Endoplásmico/metabolismo , Regulación Enzimológica de la Expresión Génica , Animales , Apoptosis , Proteínas de Unión al ADN/metabolismo , Fibroblastos/metabolismo , Ratones , Ratones Transgénicos , Mitocondrias/metabolismo , Modelos Biológicos , Factores de Transcripción del Factor Regulador X , Transducción de Señal , Factores de Transcripción/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína X Asociada a bcl-2/genéticaRESUMEN
Bax, a pro-apoptotic Bcl-2 family protein, translocates to mitochondria during apoptosis, where it causes MOMP (mitochondrial outer membrane permeabilization). MOMP releases pro-apoptotic factors, such as cytochrome c and SMAC (second mitochondrial activator of caspases)/Diablo, into the cytosol where they activate caspases. It is often inferred that Bax activation occurs in a single step, a conformational change in the protein causing its translocation and oligomerization into high-molecular-mass membrane pores. However, a number of studies have shown that Bax translocation to mitochondria does not necessarily induce MOMP. Indeed, Bax translocation can occur several hours prior to release of cytochrome c, indicating that its regulation may be a complex series of events, some of which occur following its association with mitochondria. In the present study, we have examined endogenous Bax in epithelial cells undergoing anoikis, a physiologically relevant form of apoptosis that occurs when normal cells lose contact with the ECM (extracellular matrix). Using BN-PAGE (blue native PAGE), we show that Bax forms a 200 kDa complex before caspase activation. Furthermore, Bax in this 200 kDa complex is not in the active conformation, as determined by exposure of N-terminal epitopes. These results indicate that Bax oligomerization is an event that must be interpreted differently from the currently held view that it represents the apoptotic pore.
Asunto(s)
Apoptosis/fisiología , Electroforesis en Gel de Poliacrilamida/métodos , Complejos Multiproteicos/metabolismo , Conformación Proteica , Proteína X Asociada a bcl-2/química , Proteína X Asociada a bcl-2/metabolismo , Animales , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/genética , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Caspasas/metabolismo , Adhesión Celular , Línea Celular , Reactivos de Enlaces Cruzados/metabolismo , Activación Enzimática , Células Epiteliales/citología , Células Epiteliales/fisiología , Epítopos , Humanos , Ratones , Mitocondrias/metabolismo , Peso Molecular , Complejos Multiproteicos/química , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
Unfolded proteins in the endoplasmic reticulum (ER) cause trans-autophosphorylation of the bifunctional transmembrane kinase IRE1alpha, inducing its RNase activity to splice XBP1 mRNA, in turn triggering a transcriptional program in the unfolded protein response (UPR). As we previously showed with the yeast IRE1 kinase ortholog, a single missense mutation in the ATP-binding pocket of murine IRE1alpha kinase sensitizes it to the ATP-competitive inhibitor 1NM-PP1, and subordinates RNase activity to the drug. This highly unusual mechanism of kinase signaling requiring kinase domain ligand occupancy-even through an inhibitor-to activate a nearby RNase has therefore been completely conserved through evolution. We also demonstrate that engagement of the drug-sensitized IRE1alpha kinase through this maneuver affords murine cells cytoprotection under ER stress. Thus kinase inhibitors of IRE1alpha are useful for altering the apoptotic outcome to ER stress, and could possibly be developed into drugs to treat ER stress-related diseases.