RESUMEN
Apoptosis ensures tissue homeostasis in response to developmental cues or cellular damage. Recently reported genome-wide RNAi screens have suggested that several metabolic regulators can modulate caspase activation in Drosophila. Here, we establish a previously unrecognized link between metabolism and Drosophila apoptosis by showing that cellular NADPH levels modulate the initiator caspase Dronc through its phosphorylation at S130. Depletion of NADPH removed this inhibitory phosphorylation, resulting in the activation of Dronc and subsequent cell death. Conversely, upregulation of NADPH prevented Dronc-mediated apoptosis upon DIAP1 RNAi or cycloheximide treatment. Furthermore, this CaMKII-mediated phosphorylation of Dronc hindered Dronc activation, but not its catalytic activity. Blockade of NADPH production aggravated the death-inducing activity of Dronc in specific neurons, but not in the photoreceptor cells of the eyes of transgenic flies; similarly, non-phosphorylatable Dronc was more potent than wild type in triggering specific neuronal apoptosis. Our observations reveal a novel regulatory circuitry in Drosophila apoptosis, and, as NADPH levels are elevated in cancer cells, also provide a genetic model to understand aberrations in cancer cell apoptosis resulting from metabolic alterations.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Neuronas/metabolismo , Animales , Animales Modificados Genéticamente , Western Blotting , Supervivencia Celular , Células Cultivadas , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Activación Enzimática , Inmunoprecipitación , Malatos/metabolismo , NADP/metabolismo , Neuronas/citología , ARN Interferente Pequeño/farmacologíaRESUMEN
SET domain-containing protein 2 (SETD2), a histone methyltransferase, has been identified as a target of interest in certain hematological malignancies, including multiple myeloma. This account details the discovery of EPZ-719, a novel and potent SETD2 inhibitor with a high selectivity over other histone methyltransferases. A screening campaign of the Epizyme proprietary histone methyltransferase-biased library identified potential leads based on a 2-amidoindole core. Structure-based drug design (SBDD) and drug metabolism/pharmacokinetics (DMPK) optimization resulted in EPZ-719, an attractive tool compound for the interrogation of SETD2 biology that enables in vivo target validation studies.
RESUMEN
A key challenge in the development of precision medicine is defining the phenotypic consequences of pharmacological modulation of specific target macromolecules. To address this issue, a variety of genetic, molecular and chemical tools can be used. All of these approaches can produce misleading results if the specificity of the tools is not well understood and the proper controls are not performed. In this paper we illustrate these general themes by providing detailed studies of small molecule inhibitors of the enzymatic activity of two members of the SMYD branch of the protein lysine methyltransferases, SMYD2 and SMYD3. We show that tool compounds as well as CRISPR/Cas9 fail to reproduce many of the cell proliferation findings associated with SMYD2 and SMYD3 inhibition previously obtained with RNAi based approaches and with early stage chemical probes.
Asunto(s)
Adenocarcinoma del Pulmón/tratamiento farmacológico , Carcinogénesis/genética , N-Metiltransferasa de Histona-Lisina/genética , Células A549 , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Sistemas CRISPR-Cas , Carcinogénesis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/química , Humanos , Metilación/efectos de los fármacos , Metiltransferasas/antagonistas & inhibidores , Interferencia de ARN , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
The EZH2 small-molecule inhibitor tazemetostat (EPZ-6438) is currently being evaluated in phase II clinical trials for the treatment of non-Hodgkin lymphoma (NHL). We have previously shown that EZH2 inhibitors display an antiproliferative effect in multiple preclinical models of NHL, and that models bearing gain-of-function mutations in EZH2 were consistently more sensitive to EZH2 inhibition than lymphomas with wild-type (WT) EZH2 Here, we demonstrate that cell lines bearing EZH2 mutations show a cytotoxic response, while cell lines with WT-EZH2 show a cytostatic response and only tumor growth inhibition without regression in a xenograft model. Previous work has demonstrated that cotreatment with tazemetostat and glucocorticoid receptor agonists lead to a synergistic antiproliferative effect in both mutant and wild-type backgrounds, which may provide clues to the mechanism of action of EZH2 inhibition in WT-EZH2 models. Multiple agents that inhibit the B-cell receptor pathway (e.g., ibrutinib) were found to have synergistic benefit when combined with tazemetostat in both mutant and WT-EZH2 backgrounds of diffuse large B-cell lymphomas (DLBCL). The relationship between B-cell activation and EZH2 inhibition is consistent with the proposed role of EZH2 in B-cell maturation. To further support this, we observe that cell lines treated with tazemetostat show an increase in the B-cell maturation regulator, PRDM1/BLIMP1, and gene signatures corresponding to more advanced stages of maturation. These findings suggest that EZH2 inhibition in both mutant and wild-type backgrounds leads to increased B-cell maturation and a greater dependence on B-cell activation signaling. Mol Cancer Ther; 16(11); 2586-97. ©2017 AACR.
Asunto(s)
Benzamidas/administración & dosificación , Proteína Potenciadora del Homólogo Zeste 2/genética , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Pirazoles/administración & dosificación , Piridonas/administración & dosificación , Pirimidinas/administración & dosificación , Adenina/análogos & derivados , Animales , Linfocitos B/efectos de los fármacos , Compuestos de Bifenilo , Proliferación Celular/efectos de los fármacos , Metilación de ADN/efectos de los fármacos , Sinergismo Farmacológico , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Ratones , Morfolinas , Mutación , Piperidinas , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Six fully conserved arginine residues (R129, R131, R235, R291, R319, and R340) closely grouped in the nucleotide binding site of rabbit muscle creatine kinase (rmCK) were mutated; four to alanine and all six to lysine. Kinetic analyses in the direction of phosphocreatine formation showed that all four alanine mutants led to substantial losses of activity with three (R129A, R131A, and R235A) having no detectable activity. All six lysine mutants retained variable degrees of reduced enzymatic activity. Static quenching of intrinsic tryptophan fluorescence was used to measure the binding constants for MgADP and MgATP. Nucleotide binding was at most only modestly affected by mutation of the arginine residues. Thus, the cluster of arginines seem to be primarily responsible for transition state stabilization which is further supported by the observation that none of the inactive mutants demonstrated the ability to form a transition analogue complex of MgADP.nitrate.creatine as determined by fluorescence quenching assays. As a whole, the results suggest that the most important role these residues play is to properly align the substrates for stabilization of the phosphoryl transfer reaction.
Asunto(s)
Arginina/genética , Dominio Catalítico/genética , Creatina Quinasa/genética , Mutagénesis Sitio-Dirigida , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Alanina/química , Alanina/genética , Animales , Arginina/química , Creatina/química , Creatina Quinasa/química , Creatina Quinasa/metabolismo , Forma MM de la Creatina-Quinasa , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Lisina/química , Lisina/genética , Modelos Moleculares , Mutación , Fosfocreatina/química , Unión Proteica , Conejos , Espectrometría de FluorescenciaRESUMEN
To explore the possibility that asparagine 285 plays a key role in transition state stabilization in phosphagen kinase catalysis, the N285Q, N285D, and N285A site-directed mutants of recombinant rabbit muscle creatine kinase (rmCK) were prepared and characterized. Kinetic analysis of phosphocreatine formation showed that the catalytic efficiency of each N285 mutant was reduced by approximately four orders of magnitude, with the major cause of activity loss being a reduction in k(cat) in comparison to the recombinant native CK. The data for N285Q still fit a random-order, rapid-equilibrium mechanism, with either MgATP or creatine binding first with affinities very nearly equal to those for native CK. However, the affinity for the binding of the second substrate is reduced approximately 10-fold, suggesting that addition of a single methylene group at position 285 disrupts the symphony of substrate binding. The data for the N285A mutant only fit an ordered binding mechanism, with MgATP binding first. Isosteric replacement to form the N285D mutant has almost no effect on the K(M) values for either creatine or MgATP, thus the decrease in activity is due almost entirely to a 5000-fold reduction in k(cat). Using the quenching of the intrinsic CK tryptophan fluorescence by added MgADP (Borders et al. 2002), it was found that, unlike native CK, none of the mutants have the ability to form a quaternary TSAC. We use these data to propose that asparagine 285 indeed plays a key role in transition state stabilization in the reaction catalyzed by creatine kinase and other phosphagen kinases.
Asunto(s)
Asparagina/química , Creatina Quinasa/química , Creatina/metabolismo , Isoenzimas/química , Músculo Esquelético/enzimología , Adenosina Difosfato/metabolismo , Adenosina Trifosfatasas/metabolismo , Sustitución de Aminoácidos , Animales , Sitios de Unión/fisiología , Creatina Quinasa/genética , Creatina Quinasa/metabolismo , Forma MM de la Creatina-Quinasa , Estabilidad de Enzimas , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Magnesio/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conejos , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Homeostatic maintenance of cellular mitochondria requires a dynamic balance between fission and fusion, and controlled changes in morphology are important for processes such as apoptosis and cellular division. Interphase mitochondria have been described as an interconnected network that fragments as cells enter mitosis, and this mitotic mitochondrial fragmentation is known to be regulated by the dynamin-related GTPase Drp1 (dynamin-related protein 1), a key component of the mitochondrial division machinery. Loss of Drp1 function and the subsequent failure of mitochondrial division during mitosis lead to incomplete cytokinesis and the unequal distribution of mitochondria into daughter cells. During mitotic exit and interphase, the mitochondrial network reforms. Here we demonstrate that changes in mitochondrial dynamics as cells exit mitosis are driven in part through ubiquitylation of Drp1, catalyzed by the APC/C(Cdh1) (anaphase-promoting complex/cyclosome and its coactivator Cdh1) E3 ubiquitin ligase complex. Importantly, inhibition of Cdh1-mediated Drp1 ubiquitylation and proteasomal degradation during interphase prevents the normal G1 phase regrowth of mitochondrial networks following cell division.
Asunto(s)
Cadherinas/metabolismo , Citocinesis , Mitocondrias/enzimología , Proteínas Mitocondriales/metabolismo , Mitosis , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ciclosoma-Complejo Promotor de la Anafase , Antígenos CD , Cadherinas/antagonistas & inhibidores , Cadherinas/genética , Dinaminas , Estabilidad de Enzimas , Fase G1/genética , GTP Fosfohidrolasas/deficiencia , GTP Fosfohidrolasas/genética , Expresión Génica , Silenciador del Gen , Células HEK293 , Células HeLa , Humanos , Interfase/genética , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas Asociadas a Microtúbulos/genética , Mitocondrias/genética , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Interferente Pequeño/metabolismo , Transfección , Complejos de Ubiquitina-Proteína Ligasa/antagonistas & inhibidores , Complejos de Ubiquitina-Proteína Ligasa/genética , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Morphological hallmarks of apoptosis result from activation of the caspase family of cysteine proteases, which are opposed by a pro-survival family of inhibitors of apoptosis proteins (IAPs). In Drosophila, disruption of IAP function by Reaper, HID, and Grim (RHG) proteins is sufficient to induce cell death. RHG proteins have been reported to localize to mitochondria, which, in the case of both Reaper and Grim proteins, is mediated by an amphipathic helical domain known as the GH3. Through direct binding, Reaper can bring the Drosophila IAP (DIAP1) to mitochondria, concomitantly promoting IAP auto-ubiquitination and destruction. Whether this localization is sufficient to induce DIAP1 auto-ubiquitination has not been reported. In this study we characterize the interaction between Reaper and the mitochondria using both Xenopus and Drosophila systems. We find that Reaper concentrates on the outer surface of mitochondria in a nonperipheral manner largely mediated by GH3-lipid interactions. Importantly, we show that mitochondrial targeting of DIAP1 alone is not sufficient for degradation and requires Reaper binding. Conversely, Reaper able to bind IAPs, but lacking a mitochondrial targeting GH3 domain (DeltaGH3 Reaper), can induce DIAP1 turnover only if DIAP1 is otherwise targeted to membranes. Surprisingly, targeting DIAP1 to the endoplasmic reticulum instead of mitochondria is partially effective in allowing DeltaGH3 Reaper to promote DIAP1 degradation, suggesting that co-localization of DIAP and Reaper at a membrane surface is critical for the induction of DIAP degradation. Collectively, these data provide a specific function for the GH3 domain in conferring protein-lipid interactions, demonstrate that both Reaper binding and mitochondrial localization are required for accelerated IAP degradation, and suggest that membrane localization per se contributes to DIAP1 auto-ubiquitination and degradation.
Asunto(s)
Proteínas de Drosophila/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Lípidos de la Membrana/metabolismo , Mitocondrias/metabolismo , Animales , Sitios de Unión , Western Blotting , Línea Celular , Drosophila , Proteínas de Drosophila/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Inhibidoras de la Apoptosis/genética , Liposomas/metabolismo , Microscopía Confocal , Membranas Mitocondriales/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ubiquitinación , XenopusRESUMEN
DNA-responsive checkpoints prevent cell-cycle progression following DNA damage or replication inhibition. The mitotic activator Cdc25 is suppressed by checkpoints through inhibitory phosphorylation at Ser287 (Xenopus numbering) and docking of 14-3-3. Ser287 phosphorylation is a major locus of G2/M checkpoint control, although several checkpoint-independent kinases can phosphorylate this site. We reported previously that mitotic entry requires 14-3-3 removal and Ser287 dephosphorylation. We show here that DNA-responsive checkpoints also activate PP2A/B56delta phosphatase complexes to dephosphorylate Cdc25 at a site distinct from Ser287 (T138), the phosphorylation of which is required for 14-3-3 release. However, phosphorylation of T138 is not sufficient for 14-3-3 release from Cdc25. Our data suggest that creation of a 14-3-3 "sink," consisting of phosphorylated 14-3-3 binding intermediate filament proteins, including keratins, coupled with reduced Cdc25-14-3-3 affinity, contribute to Cdc25 activation. These observations identify PP2A/B56delta as a central checkpoint effector and suggest a mechanism for controlling 14-3-3 interactions to promote mitosis.
Asunto(s)
Proteínas 14-3-3/metabolismo , Mitosis , Fosfoproteínas Fosfatasas/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Fosfatasas cdc25/metabolismo , Animales , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Replicación del ADN , Activación Enzimática , Células HCT116 , Células HeLa , Holoenzimas/metabolismo , Humanos , Filamentos Intermedios/metabolismo , Interfase , Queratinas/metabolismo , Fosforilación , Fosfotreonina/metabolismo , Proteínas Quinasas/metabolismo , Proteína Fosfatasa 2 , Subunidades de Proteína/metabolismoRESUMEN
Bcl-2 has been described both as an inhibitor of programmed cell death and as an inhibitor of mitochondrial dysfunction during apoptosis. It is still not clear what biochemical activity of Bcl-2 is responsible for its function, but increasing evidence indicates that a functional activity of Bcl-2 on the endoplasmic reticulum (ER) protects mitochondria under diverse circumstances. Indeed, an emerging hypothesis is that, during apoptosis, the Bcl-2 family regulates ER-to-mitochondrion communication by BH3-only proteins and calcium ions and thereby triggers mitochondrial dysfunction and cell death.
Asunto(s)
Apoptosis/fisiología , Retículo Endoplásmico/metabolismo , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Caspasas/metabolismo , Células Cultivadas , Citocromos c/metabolismo , Humanos , Proteína bcl-XRESUMEN
Bcl-2 family members have been shown to be key mediators of apoptosis as either pro- or anti-apoptotic factors. It is thought that both classes of Bcl-2 family members act at the level of the mitochondria to regulate apoptosis, although the founding anti-apoptotic family member, Bcl-2 is localized to the endoplasmic reticulum (ER), mitochondrial, and nuclear membranes. In order to better understand the effect of Bcl-2 localization on its activity, we have utilized a Bcl-2 mutant that localizes only to the ER membrane, designated Bcl-2Cb5. Bcl-2Cb5 was expressed in MDA-MB-468 cells, which protected against apoptosis induced by the kinase inhibitor, staurosporine. Data presented here show that Bcl-2Cb5 inhibits this process by blocking Bax activation and cytochrome c release. Furthermore, we show that Bcl-2Cb5 can inhibit the activation of a constitutively mitochondrial mutant of Bax, indicating that an intermediate between Bcl-2 on the ER and Bax on the mitochondria must exist. We demonstrate that this intermediate is likely a BH3-only subfamily member. Data presented here show that Bcl-2Cb5 can sequester a constitutively active form of Bad (Bad3A) from the mitochondria and prevent it from activating Bax. These data suggest that Bcl-2 indirectly protects mitochondrial membranes from Bax, via BH3-only proteins.
Asunto(s)
Retículo Endoplásmico/fisiología , Mitocondrias/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Apoptosis , Sitios de Unión , Neoplasias de la Mama , Femenino , Genes bcl-2 , Humanos , Membrana Nuclear/fisiología , Proteínas Recombinantes/metabolismo , Transfección , Células Tumorales Cultivadas , Proteína X Asociada a bcl-2RESUMEN
Glucocorticoid hormones induce apoptosis in lymphoid cells. This process requires de novo RNA/protein synthesis. Here we report the identification and cloning of a novel dexamethasone-induced gene designated dig2. Using Affymetrix oligonucleotide microarray analysis of approximately 10,000 genes and expressed sequence tags, we found that the expression of dig2 mRNA is significantly induced not only in the murine T cell lymphoma lines S49.A2 and WEHI7.2 but also in normal mouse thymocytes following dexamethasone treatment. This result was confirmed by Northern blot analysis. The induction of dig2 mRNA by dexamethasone appears to be mediated through the glucocorticoid receptor as it is blocked in the presence of RU486, a glucocorticoid receptor antagonist. Furthermore, we demonstrated that dig2 is a novel stress response gene, as its mRNA is induced in response to a variety of cellular stressors including thapsigargin, tunicamycin, and heat shock. In addition, the levels of dig2 mRNA were up-regulated after treatment with the apoptosis-inducing chemotherapeutic drug etoposide. Though the function of dig2 is unknown, dig2 appears to have a pro-survival function, as overexpression of dig2 reduces the sensitivity of WEHI7.2 cells to dexamethasone-induced apoptosis.