RESUMEN
RIPK2 mediates pro-inflammatory signaling from the bacterial sensors NOD1 and NOD2, and is an emerging therapeutic target in autoimmune and inflammatory diseases. We observed that cellular RIPK2 can be potently inhibited by type II inhibitors that displace the kinase activation segment, whereas ATP-competitive type I inhibition was only poorly effective. The most potent RIPK2 inhibitors were the US Food and Drug Administration-approved drugs ponatinib and regorafenib. Their mechanism of action was independent of NOD2 interaction and involved loss of downstream kinase activation as evidenced by lack of RIPK2 autophosphorylation. Notably, these molecules also blocked RIPK2 ubiquitination and, consequently, inflammatory nuclear factor κB signaling. In monocytes, the inhibitors selectively blocked NOD-dependent tumor necrosis factor production without affecting lipopolysaccharide-dependent pathways. We also determined the first crystal structure of RIPK2 bound to ponatinib, and identified an allosteric site for inhibitor development. These results highlight the potential for type II inhibitors to treat indications of RIPK2 activation as well as inflammation-associated cancers.
Asunto(s)
Proteína Adaptadora de Señalización NOD1/antagonistas & inhibidores , Proteína Adaptadora de Señalización NOD2/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Células Cultivadas , Humanos , Imidazoles/química , Imidazoles/farmacología , Inflamación/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Unión Proteica , Inhibidores de Proteínas Quinasas/química , Piridazinas/química , Piridazinas/farmacología , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , Células Sf9 , Transducción de Señal/efectos de los fármacos , Ubiquitinación/efectos de los fármacosRESUMEN
RIPK1 and RIPK3, two closely related RIPK family members, have emerged as important regulators of pathologic cell death and inflammation. In the current work, we report that the Bcr-Abl inhibitor and anti-leukemia agent ponatinib is also a first-in-class dual inhibitor of RIPK1 and RIPK3. Ponatinib potently inhibited multiple paradigms of RIPK1- and RIPK3-dependent cell death and inflammatory tumor necrosis factor alpha (TNF-α) gene transcription. We further describe design strategies that utilize the ponatinib scaffold to develop two classes of inhibitors (CS and PN series), each with greatly improved selectivity for RIPK1. In particular, we detail the development of PN10, a highly potent and selective "hybrid" RIPK1 inhibitor, capturing the best properties of two different allosteric RIPK1 inhibitors, ponatinib and necrostatin-1. Finally, we show that RIPK1 inhibitors from both classes are powerful blockers of TNF-induced injury in vivo. Altogether, these findings outline promising candidate molecules and design approaches for targeting RIPK1- and RIPK3-driven inflammatory pathologies.
Asunto(s)
Antineoplásicos/farmacología , Imidazoles/farmacología , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas/farmacología , Piridazinas/farmacología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/química , Secuencia de Aminoácidos , Animales , Antineoplásicos/química , Femenino , Células HEK293 , Humanos , Imidazoles/química , Células Jurkat , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/química , Piridazinas/química , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Especificidad por SustratoRESUMEN
Necrosis is a primary form of cell death in a variety of human pathologies. The deleterious nature of necrosis, including its propensity to promote inflammation, and the relative lack of the cells displaying necrotic morphology under physiologic settings, such as during development, have contributed to the notion that necrosis represents a form of pathologic stress-induced nonspecific cell lysis. However, this notion has been challenged in recent years by the discovery of a highly regulated form of necrosis, termed regulated necrosis or necroptosis. Necroptosis is now recognized by the work of multiple labs, as an important, drug-targetable contributor to necrotic injury in many pathologies, including ischemia-reperfusion injuries (heart, brain, kidney, liver), brain trauma, eye diseases, and acute inflammatory conditions. In this review, we describe the methods to analyze cellular necroptosis and activity of its key mediator, RIP1 kinase.
Asunto(s)
Bioensayo/métodos , Necrosis/enzimología , Proteínas de Complejo Poro Nuclear/biosíntesis , Proteínas de Unión al ARN/biosíntesis , Daño por Reperfusión/enzimología , Apoptosis/genética , Humanos , Proteínas de Complejo Poro Nuclear/genética , Proteínas Quinasas/genética , Proteínas de Unión al ARN/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Daño por Reperfusión/genéticaRESUMEN
Interferons (IFNs) are cytokines with powerful immunomodulatory and antiviral properties, but less is known about how they induce cell death. Here, we show that both type I (α/ß) and type II (γ) IFNs induce precipitous receptor-interacting protein (RIP)1/RIP3 kinase-mediated necrosis when the adaptor protein Fas-associated death domain (FADD) is lost or disabled by phosphorylation, or when caspases (e.g., caspase 8) are inactivated. IFN-induced necrosis proceeds via progressive assembly of a RIP1-RIP3 "necrosome" complex that requires Jak1/STAT1-dependent transcription, but does not need the kinase activity of RIP1. Instead, IFNs transcriptionally activate the RNA-responsive protein kinase PKR, which then interacts with RIP1 to initiate necrosome formation and trigger necrosis. Although IFNs are powerful activators of necrosis when FADD is absent, these cytokines are likely not the dominant inducers of RIP kinase-driven embryonic lethality in FADD-deficient mice. We also identify phosphorylation on serine 191 as a mechanism that disables FADD and collaborates with caspase inactivation to allow IFN-activated necrosis. Collectively, these findings outline a mechanism of IFN-induced RIP kinase-dependent necrotic cell death and identify FADD and caspases as negative regulators of this process.
Asunto(s)
Puntos de Control del Ciclo Celular/fisiología , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Interferón gamma/metabolismo , Modelos Moleculares , Necrosis/metabolismo , Transducción de Señal/fisiología , Animales , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Proteína de Dominio de Muerte Asociada a Fas/química , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteínas Activadoras de GTPasa/metabolismo , Inmunoprecipitación , Ratones , Ratones Noqueados , Fosforilación , Interferencia de ARN , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Factor de Transcripción STAT1/metabolismo , eIF-2 Quinasa/metabolismoRESUMEN
Necroptosis is a novel form of regulated non-apoptotic cell death, which displays morphological features of necrosis. The kinase activity of receptor-interacting protein kinase 1 (RIP1) is a critical component in signaling for necroptosis. The development of assays to evaluate RIP1 kinase activity is important in the further development of existing and novel inhibitors of necroptosis. Here, we describe RIP1 protein expression and purification from mammalian and insect cells as well as two in vitro kinase assays to detect RIP1 kinase activity and inhibition.
Asunto(s)
Pruebas de Enzimas/métodos , Necrosis/patología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Células HEK293 , Humanos , Imidazoles/química , Imidazoles/metabolismo , Indoles/química , Indoles/metabolismo , Radiometría , Proteína Serina-Treonina Quinasas de Interacción con Receptores/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Células Sf9RESUMEN
Receptor Interacting Protein 1 (RIP1) kinase is one of the key mediators of tumor necrosis factor alpha (TNF-α) signaling and is critical for activation of necroptotic cell death. We developed a method for expression of recombinant kinase, utilizing baculovirus co-infection of Cdc37, an Hsp90 co-chaperone, and RIP1-His, followed by a two-step purification scheme. After optimization, 1-3mg of highly purified RIP1 kinase was typically obtained from a 1L of Sf9 cells. The recombinant protein displayed kinase activity that was blocked by RIP1 inhibitors, necrostatins. The purified protein was used to develop a simple and robust thermal shift assay for further assessment of RIP1 inhibitors.
Asunto(s)
Baculoviridae/genética , Clonación Molecular , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Animales , Línea Celular , Humanos , Imidazoles/farmacología , Indoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Proteína Serina-Treonina Quinasas de Interacción con Receptores/aislamiento & purificación , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Necrotic cell death is prevalent in many different pathological disease states and in traumatic injury. Necroptosis is a form of necrosis that stems from specific signaling pathways, with the key regulator being receptor interacting protein 1 (RIP1), a serine/threonine kinase. Specific inhibitors of RIP1, termed necrostatins, are potent inhibitors of necroptosis. Necrostatins are structurally distinct from one another yet still possess the ability to inhibit RIP1 kinase activity. To further understand the differences in the binding of the various necrostatins to RIP1 and to develop a robust high-throughput screening (HTS) assay, which can be used to identify new classes of RIP1 inhibitors, we synthesized fluorescein derivatives of Necrostatin-1 (Nec-1) and Nec-3. These compounds were used to establish a fluorescence polarization (FP) assay to directly measure the binding of necrostatins to RIP1 kinase. The fluorescein-labeled compounds are well suited for HTS because the assays have a dimethyl sulfoxide (DMSO) tolerance up to 5% and Z' scores of 0.62 (fluorescein-Nec-1) and 0.57 (fluorescein-Nec-3). In addition, results obtained from the FP assays and ligand docking studies provide insights into the putative binding sites of Nec-1, Nec-3, and Nec-4.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento , Imidazoles/química , Indoles/química , Inhibidores de Proteínas Quinasas/análogos & derivados , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Baculoviridae , Sitios de Unión , Unión Competitiva , Línea Celular , Fluoresceína , Polarización de Fluorescencia , Humanos , Imidazoles/farmacología , Indoles/farmacología , Cinética , Ligandos , Modelos Moleculares , Necrosis/prevención & control , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/química , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Spodoptera , Coloración y EtiquetadoRESUMEN
The SecA molecular nanomachine in bacteria uses energy from ATP hydrolysis to drive post-translational secretion of preproteins through the SecYEG translocon. Cytosolic SecA exists in a dimeric, "closed" state with relatively low ATPase activity. After binding to the translocon, SecA undergoes major conformational rearrangement, leading to a state that is structurally more "open", has elevated ATPase activity, and is active in translocation. The structural details underlying this conformational change in SecA remain incompletely defined. Most SecA crystal structures report on the cytosolic form; only one structure sheds light on a form of SecA that has engaged the translocon. We have used mild destabilization of SecA to trigger conformational changes that mimic those in translocation-active SecA and thus study its structural changes in a simplified, soluble system. Results from circular dichroism, tryptophan fluorescence, and limited proteolysis demonstrate that the SecA conformational reorganization involves disruption of several domain-domain interfaces, partial unfolding of the second nucleotide binding fold (NBF) II, partial dissociation of the helical scaffold domain (HSD) from NBF I and II, and restructuring of the 30 kDa C-terminal region. These changes account for the observed high translocation SecA ATPase activity because they lead to the release of an inhibitory C-terminal segment (called intramolecular regulator of ATPase 1, or IRA1) and of constraints on NBF II (or IRA2) that allow it to stimulate ATPase activity. The observed conformational changes thus position SecA for productive interaction with the SecYEG translocon and for transfer of segments of its passenger protein across the translocon.
Asunto(s)
Adenosina Trifosfatasas/química , Proteínas Bacterianas/química , Proteínas de Transporte de Membrana/química , Adenosina Trifosfato/química , Bacillus subtilis/metabolismo , Benzofenonas/farmacología , Reactivos de Enlaces Cruzados/farmacología , Citosol/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Hidrólisis , Maleimidas/farmacología , Proteínas de la Membrana/metabolismo , Conformación Proteica , Desnaturalización Proteica , Señales de Clasificación de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , Canales de Translocación SEC , Proteína SecA , Espectrometría de Fluorescencia/métodos , Triptófano/químicaRESUMEN
The information for correct localization of newly synthesized proteins in both prokaryotes and eukaryotes resides in self-contained, often transportable targeting sequences. Of these, signal sequences specify that a protein should be secreted from a cell or incorporated into the cytoplasmic membrane. A central puzzle is presented by the lack of primary structural homology among signal sequences, although they share common features in their sequences. Synthetic signal peptides have enabled a wide range of studies of how these "zipcodes" for protein secretion are decoded and used to target proteins to the protein machinery that facilitates their translocation across and integration into membranes. We review research on how the information in signal sequences enables their passenger proteins to be correctly and efficiently localized. Synthetic signal peptides have made possible binding and crosslinking studies to explore how selectivity is achieved in recognition by the signal sequence-binding receptors, signal recognition particle, or SRP, which functions in all organisms, and SecA, which functions in prokaryotes and some organelles of prokaryotic origins. While progress has been made, the absence of atomic resolution structures for complexes of signal peptides and their receptors has definitely left many questions to be answered in the future.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Señales de Clasificación de Proteína , Partícula de Reconocimiento de Señal/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Reactivos de Enlaces Cruzados/química , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Transporte de Proteínas , Canales de Translocación SEC , Proteína SecA , Partícula de Reconocimiento de Señal/química , Partícula de Reconocimiento de Señal/genéticaRESUMEN
At low concentrations, hydrogen peroxide (H(2)O(2)) is a positive endogenous regulator of mammalian cell proliferation and survival; however, the signal transduction pathways involved in these processes are poorly understood. In primary human endothelial cells, low concentrations of H(2)O(2) stimulated the rapid phosphorylation of the acidic C-terminal domain (ACD) of heterogeneous nuclear ribonucleoprotein C (hnRNP-C), a nuclear restricted pre-mRNA-binding protein, at Ser(240) and at Ser(225)-Ser(228). A kinase activity was identified in mouse liver that phosphorylates the ACD of hnRNP-C at Ser(240) and at two sites at Ser(225)-Ser(228). The kinase was purified and identified by tandem mass spectrometry as protein kinase CK1alpha (formerly casein kinase 1alpha). Protein kinase CK1alpha immunoprecipitated from primary human endothelial cell nuclei also phosphorylated the ACD of hnRNP-C at these positions. Pretreatment of endothelial cells with the protein kinase CK1-specific inhibitor IC261 prevented the H(2)O(2)-stimulated phosphorylation of hnRNP-C. Utilizing phosphoserine-mimicking Ser-to-Glu point mutations, the effects of phosphorylation on hnRNP-C function were investigated by quantitative equilibrium fluorescence RNA binding analyses. Wild-type hnRNP-C1 and hnRNP-C1 modified at the basal sites of phosphorylation (S247E and S286E) both avidly bound RNA with similar binding constants. In contrast, hnRNP-C1 that was also modified at the CK1alpha phosphorylation sites exhibited a 14-500-fold decrease in binding affinity, demonstrating that CK1alpha-mediated phosphorylation modulates the mRNA binding ability of hnRNP-C.
Asunto(s)
Caseína Quinasa Ialfa/fisiología , Ribonucleoproteína Heterogénea-Nuclear Grupo C/metabolismo , Peróxido de Hidrógeno/farmacología , Floroglucinol/análogos & derivados , ARN Mensajero/metabolismo , Secuencia de Aminoácidos , Animales , Caseína Quinasa Ialfa/metabolismo , Células Cultivadas , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Electroforesis en Gel Bidimensional , Endotelio Vascular/citología , Escherichia coli/metabolismo , Evolución Molecular , Humanos , Inmunoprecipitación , Indoles/farmacología , Cinética , Hígado/metabolismo , Ratones , Datos de Secuencia Molecular , Floroglucinol/farmacología , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , ARN/metabolismo , Homología de Secuencia de Aminoácido , Serina/química , Espectrometría de FluorescenciaRESUMEN
Retroviral assembly is driven by multiple interactions mediated by the Gag polyprotein, the main structural component of the forming viral shell. Critical determinants of Gag oligomerization are contained within the C-terminal domain (CTD) of the capsid protein, which also harbors a conserved sequence motif, the major homology region (MHR), in the otherwise highly variable Gag. An unexpected clue about the MHR function in retroviral assembly emerges from the structure of the zinc finger-associated SCAN domain we describe here. The SCAN dimer adopts a fold almost identical to that of the retroviral capsid CTD but uses an entirely different dimerization interface caused by swapping the MHR-like element between the monomers. Mutations in retroviral capsid proteins and functional data suggest that a SCAN-like MHR-swapped CTD dimer forms during immature particle assembly. In the SCAN-like dimer, the MHR contributes the major part of the large intertwined dimer interface explaining its functional significance.
Asunto(s)
Proteínas de la Cápside/química , VIH-1/química , Proteínas Represoras/química , Secuencia de Aminoácidos , Proteínas de la Cápside/genética , Dimerización , VIH-1/genética , VIH-1/fisiología , Humanos , Técnicas In Vitro , Factores de Transcripción de Tipo Kruppel , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Resonancia Magnética Nuclear Biomolecular , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Represoras/genética , Homología de Secuencia de Aminoácido , Ensamble de Virus , Dedos de Zinc/genéticaRESUMEN
The SCAN domain is a highly conserved dimerization motif that is vertebrate-specific and found near the N-terminus of C(2)H(2) zinc finger proteins (SCAN-ZFP). Although the function of most SCAN-ZFPs is unknown, some have been implicated in the transcriptional regulation of growth factors, genes involved in lipid metabolism, as well as other genes involved in cell survival and differentiation. Here we utilize a bioinformatics approach to define the structures and gene locations of the 71 members of the human SCAN domain family, as well as to assess the conserved syntenic segments in the mouse genome and identify potential orthologs. The genes encoding SCAN domains are clustered, often in tandem arrays, in both the human and mouse genomes and are capable of generating isoforms that may affect the function of family members. Twenty-three members of the mouse SCAN family appear to be orthologous with human family members, and human-specific cluster expansions were observed. Remarkably, the SCAN domains in lower vertebrates are not associated with C(2)H(2) zinc finger genes, but are contained in large retrovirus-like polyproteins. Collectively, these studies define a large family of vertebrate-specific transcriptional regulators that may have rapidly expanded during recent evolution.
Asunto(s)
Factores de Transcripción/genética , Dedos de Zinc/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Mapeo Cromosómico , Secuencia Conservada/genética , Bases de Datos Genéticas , Expresión Génica , Genes/genética , Genoma Humano , Humanos , Ratones , Datos de Secuencia Molecular , Filogenia , Homología de Secuencia de Aminoácido , Vertebrados/genéticaRESUMEN
Hydrogen peroxide (H2O2) is a recently recognized second messenger, which regulates mammalian cell proliferation and migration. The biochemical mechanisms by which mammalian cells sense and respond to low concentrations of H2O2 are poorly understood. Recently, heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP-C1/C2) was found to be rapidly phosphorylated in response to the application of low concentrations of H2O2 to human endothelial cells. Here, using tandem mass spectrometry, four sites of phosphorylation are identified in hnRNP-C1/C2, all of which are in the acidic C-terminal domain of the protein. Under resting conditions, the protein is phosphorylated at S247 and S286. In response to low concentrations of H2O2, there is increased phosphorylation at S240 and at one of the four contiguous serine residues from S225-S228. Studies using a recombinant acidic C-terminal domain of hnRNP-C overexpressed in Escherichia coli demonstrate that protein kinase CK2 phosphorylates hnRNP-C1/C2 at S247, while protein kinase A and several protein kinase C isoforms fail to phosphorylate the isolated domain. These findings demonstrate that the acidic C-terminal domain of hnRNP-C1/C2 serves as the site for both basal and stimulated phosphorylation, indicating that this domain may play an important role in the regulation of mRNA binding by hnRNP-C1/C2.
Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo C/metabolismo , Peróxido de Hidrógeno/farmacología , Secuencia de Aminoácidos , Animales , Células CHO , Línea Celular , Pollos , Cricetinae , Células HeLa , Ribonucleoproteína Heterogénea-Nuclear Grupo C/análisis , Ribonucleoproteína Heterogénea-Nuclear Grupo C/genética , Humanos , Concentración de Iones de Hidrógeno , Células K562 , Datos de Secuencia Molecular , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosforilación/efectos de los fármacos , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes de Fusión/fisiología , Espectrometría de Masa por Ionización de Electrospray , Células Tumorales CultivadasRESUMEN
The SCAN domain is a conserved region of 84 residues found predominantly in zinc finger DNA-binding proteins in vertebrates. The SCAN domain appears to control the association of SCAN domain containing proteins into noncovalent complexes and may be the primary mechanism underlying partner choice in the oligomerization of these transcription factors. Here we have overexpressed, purified, and characterized the isolated SCAN domain (amino acids 37-132) from ZNF174. Both size exclusion chromatography and equilibrium sedimentation analysis demonstrate that the ZNF174 SCAN domain forms a homodimer. Circular dichroism shows that the isolated SCAN domain dimer has approximately 42% alpha-helix. Thermal denaturation experiments indicate that the SCAN domain undergoes a single reversible unfolding transition with a T(m) of over 70 degrees C. The midpoint of the equilibrium unfolding transition increases with increasing protein concentration, consistent with a two-state unfolding transition in which folded dimer is in equilibrium with unfolded monomer. These findings demonstrate that the isolated SCAN domain forms a stable dimer and support a model in which the SCAN domain is capable of mediating the selective dimerization of a large family of vertebrate-specific, zinc finger-containing transcription factors.