RESUMEN
Deubiquitinating enzymes (DUBs) are an emerging drug target class of ~100 proteases that cleave ubiquitin from protein substrates to regulate many cellular processes. A lack of selective chemical probes impedes pharmacologic interrogation of this important gene family. DUBs engage their cognate ligands through a myriad of interactions. We embrace this structural complexity to tailor a chemical diversification strategy for a DUB-focused covalent library. Pairing our library with activity-based protein profiling as a high-density primary screen, we identify selective hits against 23 endogenous DUBs spanning four subfamilies. Optimization of an azetidine hit yields a probe for the understudied DUB VCPIP1 with nanomolar potency and in-family selectivity. Our success in identifying good chemical starting points as well as structure-activity relationships across the gene family from a modest but purpose-build library challenges current paradigms that emphasize ultrahigh throughput in vitro or virtual screens against an ever-increasing scope of chemical space.
Asunto(s)
Endopeptidasas , Ubiquitina , Ubiquitina/metabolismo , Endopeptidasas/metabolismo , Péptido Hidrolasas/metabolismo , Relación Estructura-Actividad , Enzimas Desubicuitinizantes/metabolismo , UbiquitinaciónRESUMEN
Deleted in Breast Cancer 1 (DBC1) is an important metabolic sensor. Previous studies have implicated DBC1 in various cellular functions, notably cell proliferation, apoptosis, histone modification, and adipogenesis. However, current reports about the role of DBC1 in tumorigenesis are controversial and designate DBC1 alternatively as a tumor suppressor or a tumor promoter. In the present study, we report that polyoma small T antigen (PyST) associates with DBC1 in mammalian cells, and this interaction leads to the posttranslational downregulation of DBC1 protein levels. When coexpressed, DBC1 overcomes PyST-induced mitotic arrest and promotes the exit of cells from mitosis. Using both transient and stable modes of PyST expression, we also show that cellular DBC1 is subjected to degradation by LKB1, a tumor suppressor and cellular energy sensor kinase, in an AMP kinase-independent manner. Moreover, LKB1 negatively regulates the phosphorylation as well as activity of the prosurvival kinase AKT1 through DBC1 and its downstream pseudokinase substrate, Tribbles 3 (TRB3). Using both transient transfection and stable cell line approaches as well as soft agar assay, we demonstrate that DBC1 has oncogenic potential. In conclusion, our study provides insight into a novel signaling axis that connects LKB1, DBC1, TRB3, and AKT1. We propose that the LKB1-DBC1-AKT1 signaling paradigm may have an important role in the regulation of cell cycle and apoptosis and consequently tumorigenesis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Antígenos Virales de Tumores , Proteínas de Ciclo Celular , Proteínas del Tejido Nervioso , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis , Carcinogénesis , Proteínas de Ciclo Celular/metabolismo , Regulación hacia Abajo , Humanos , Proteínas del Tejido Nervioso/metabolismo , Sirtuina 1/metabolismoRESUMEN
Parallel reaction monitoring (PRM) has emerged as a popular approach for targeted protein quantification. With high ion utilization efficiency and first-in-class acquisition speed, the timsTOF Pro provides a powerful platform for PRM analysis. However, sporadic chromatographic drift in peptide retention time represents a fundamental limitation for the reproducible multiplexing of targets across PRM acquisitions. Here, we present PRM-LIVE, an extensible, Python-based acquisition engine for the timsTOF Pro, which dynamically adjusts detection windows for reproducible target scheduling. In this initial implementation, we used iRT peptides as retention time standards and demonstrated reproducible detection and quantification of 1857 tryptic peptides from the cell lysate in a 60 min PRM-LIVE acquisition. As an application in functional proteomics, we use PRM-LIVE in an activity-based protein profiling platform to assess binding selectivity of small-molecule inhibitors against 220 endogenous human kinases.
Asunto(s)
Espectrometría de Movilidad Iónica , Proteómica , Humanos , Espectrometría de Masas , Péptidos , ProteínasRESUMEN
The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Peptidilprolil Isomerasa de Interacción con NIMA/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Complex neural circuitry requires stable connections formed by lengthy axons. To maintain these functional circuits, fast transport delivers RNAs to distal axons where they undergo local translation. However, the mechanism that enables long-distance transport of RNA granules is not yet understood. Here, we demonstrate that a complex containing RNA and the RNA-binding protein (RBP) SFPQ interacts selectively with a tetrameric kinesin containing the adaptor KLC1 and the motor KIF5A. We show that the binding of SFPQ to the KIF5A/KLC1 motor complex is required for axon survival and is impacted by KIF5A mutations that cause Charcot-Marie Tooth (CMT) disease. Moreover, therapeutic approaches that bypass the need for local translation of SFPQ-bound proteins prevent axon degeneration in CMT models. Collectively, these observations indicate that KIF5A-mediated SFPQ-RNA granule transport may be a key function disrupted in KIF5A-linked neurologic diseases and that replacing axonally translated proteins serves as a therapeutic approach to axonal degenerative disorders.
Asunto(s)
Transporte Axonal , Axones/metabolismo , Cinesinas/metabolismo , Factor de Empalme Asociado a PTB/metabolismo , ARN/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Gránulos Citoplasmáticos/metabolismo , Ganglios Espinales/metabolismo , Células HEK293 , Humanos , Proteínas Asociadas a Microtúbulos , Mitocondrias/metabolismo , Mutación/genética , Péptidos/metabolismo , Fosforilación , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Células Receptoras Sensoriales/metabolismoRESUMEN
Notch signaling regulates squamous cell proliferation and differentiation and is frequently disrupted in squamous cell carcinomas, in which Notch is tumor suppressive. Here, we show that conditional activation of Notch in squamous cells activates a context-specific gene expression program through lineage-specific regulatory elements. Among direct Notch target genes are multiple DNA damage response genes, including IER5, which we show is required for Notch-induced differentiation of squamous carcinoma cells and TERT-immortalized keratinocytes. IER5 is epistatic to PPP2R2A, a gene that encodes the PP2A B55α subunit, which we show interacts with IER5 in cells and in purified systems. Thus, Notch and DNA-damage response pathways converge in squamous cells on common genes that promote differentiation, which may serve to eliminate damaged cells from the proliferative pool. We further propose that crosstalk involving Notch and PP2A enables tuning and integration of Notch signaling with other pathways that regulate squamous differentiation.
Asunto(s)
Diferenciación Celular/genética , Células Epiteliales/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Nucleares/metabolismo , Receptores Notch/metabolismo , Carcinoma de Células Escamosas/metabolismo , Línea Celular , Daño del ADN/genética , Humanos , Proteínas Inmediatas-Precoces/genética , Queratinocitos/metabolismo , Proteínas Nucleares/genética , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Receptores Notch/genética , Transducción de Señal/genéticaRESUMEN
DNA double-strand breaks (DSBs) are repaired through homology-directed repair (HDR) or non-homologous end joining (NHEJ). BRCA1/2-deficient cancer cells cannot perform HDR, conferring sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPi). However, concomitant loss of the pro-NHEJ factors 53BP1, RIF1, REV7-Shieldin (SHLD1-3) or CST-DNA polymerase alpha (Pol-α) in BRCA1-deficient cells restores HDR and PARPi resistance. Here, we identify the TRIP13 ATPase as a negative regulator of REV7. We show that REV7 exists in active 'closed' and inactive 'open' conformations, and TRIP13 catalyses the inactivating conformational change, thereby dissociating REV7-Shieldin to promote HDR. TRIP13 similarly disassembles the REV7-REV3 translesion synthesis (TLS) complex, a component of the Fanconi anaemia pathway, inhibiting error-prone replicative lesion bypass and interstrand crosslink repair. Importantly, TRIP13 overexpression is common in BRCA1-deficient cancers, confers PARPi resistance and correlates with poor prognosis. Thus, TRIP13 emerges as an important regulator of DNA repair pathway choice-promoting HDR, while suppressing NHEJ and TLS.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/genética , Proteína BRCA1/deficiencia , Proteínas de Ciclo Celular/genética , Reparación del ADN/genética , Reparación del ADN por Recombinación/genética , ATPasas Asociadas con Actividades Celulares Diversas/efectos de los fármacos , Proteínas de Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Daño del ADN/efectos de los fármacos , Reparación del ADN por Unión de Extremidades/genética , Reparación del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Replicación del ADN/genética , Humanos , Proteínas Mad2/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Proteínas de Unión a Telómeros/efectos de los fármacos , Proteínas de Unión a Telómeros/genéticaRESUMEN
Alterations involving serine-threonine phosphatase PP2A subunits occur in a range of human cancers, and partial loss of PP2A function contributes to cell transformation. Displacement of regulatory B subunits by the SV40 Small T antigen (ST) or mutation/deletion of PP2A subunits alters the abundance and types of PP2A complexes in cells, leading to transformation. Here, we show that ST not only displaces common PP2A B subunits but also promotes A-C subunit interactions with alternative B subunits (B''', striatins) that are components of the Striatin-interacting phosphatase and kinase (STRIPAK) complex. We found that STRN4, a member of STRIPAK, is associated with ST and is required for ST-PP2A-induced cell transformation. ST recruitment of STRIPAK facilitates PP2A-mediated dephosphorylation of MAP4K4 and induces cell transformation through the activation of the Hippo pathway effector YAP1. These observations identify an unanticipated role of MAP4K4 in transformation and show that the STRIPAK complex regulates PP2A specificity and activity.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Unión a Calmodulina/genética , Proteínas de Unión a Calmodulina/metabolismo , Proliferación Celular , Femenino , Técnicas de Silenciamiento del Gen , Células HEK293 , Xenoinjertos , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Fosfoproteínas Fosfatasas/genética , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAPRESUMEN
Canonical Notch signaling relies on regulated proteolysis of the receptor Notch to generate a nuclear effector that induces the transcription of Notch-responsive genes. In higher organisms, one Notch-responsive gene that is activated in many different cell types encodes the Notch-regulated ankyrin repeat protein (NRARP), which acts as a negative feedback regulator of Notch responses. Here, we showed that NRARP inhibited the growth of Notch-dependent T cell acute lymphoblastic leukemia (T-ALL) cell lines and bound directly to the core Notch transcriptional activation complex (NTC), requiring both the transcription factor RBPJ and the Notch intracellular domain (NICD), but not Mastermind-like proteins or DNA. The crystal structure of an NRARP-NICD1-RBPJ-DNA complex, determined to 3.75 Å resolution, revealed that the assembly of NRARP-NICD1-RBPJ complexes relied on simultaneous engagement of RBPJ and NICD1, with the three ankyrin repeats of NRARP extending the Notch1 ankyrin repeat stack. Mutations at the NRARP-NICD1 interface disrupted entry of the proteins into NTCs and abrogated feedback inhibition in Notch signaling assays in cultured cells. Forced expression of NRARP reduced the abundance of NICD in cells, suggesting that NRARP may promote the degradation of NICD. These studies establish the structural basis for NTC engagement by NRARP and provide insights into a critical negative feedback mechanism that regulates Notch signaling.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Células Jurkat , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Mutación , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Estructura Cuaternaria de Proteína , Receptores Notch/química , Receptores Notch/genéticaRESUMEN
Mitotic cells attenuate the DNA damage response (DDR) by phosphorylating 53BP1, a critical DDR mediator, to prevent its localization to damaged chromatin. Timely dephosphorylation of 53BP1 is critical for genome integrity, as premature recruitment of 53BP1 to DNA lesions impairs mitotic fidelity. Protein phosphatase 4 (PP4) dephosphorylates 53BP1 in late mitosis to allow its recruitment to DNA lesions in G1. How cells appropriately dephosphorylate 53BP1, thereby restoring DDR, is unclear. Here, we elucidate the underlying mechanism of kinetic control of 53BP1 dephosphorylation in mitosis. We demonstrate that CDK5, a kinase primarily functional in post-mitotic neurons, is active in late mitotic phases in non-neuronal cells and directly phosphorylates PP4R3ß, the PP4 regulatory subunit that recognizes 53BP1. Specific inhibition of CDK5 in mitosis abrogates PP4R3ß phosphorylation and abolishes its recognition and dephosphorylation of 53BP1, ultimately preventing the localization of 53BP1 to damaged chromatin. Our results establish CDK5 as a regulator of 53BP1 recruitment.
Asunto(s)
Quinasa 5 Dependiente de la Ciclina/metabolismo , Reparación del ADN/genética , Fase G1/genética , Fosfoproteínas Fosfatasas/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Línea Celular Tumoral , Daño del ADN/genética , Células HEK293 , Células HeLa , Humanos , Mitosis/genética , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteína 1 de Unión al Supresor Tumoral P53/genéticaRESUMEN
Affinity purification followed by mass spectrometry has become the technique of choice to identify binding partners in biochemical complexes isolated from a physiologic cellular context. In this report we detail our protocol for tandem affinity purification (TAP) primarily based on the use of the FLAG and HA peptide epitopes, with a particular emphasis on factors affecting yield and specificity, as well as steps to implement an automated version of the TAP procedure. © 2019 by John Wiley & Sons, Inc.
Asunto(s)
Retroviridae/aislamiento & purificación , Purificación por Afinidad en Tándem/métodos , Espectrometría de Masas en Tándem/métodos , Células HEK293 , Células HeLa , Humanos , Oligopéptidos/química , Proteómica , Retroviridae/química , Retroviridae/genéticaRESUMEN
Reactive oxygen species (ROS) are a byproduct of cell metabolism, and can also arise from environmental sources, such as toxins or radiation. Depending on dose and context, ROS have both beneficial and deleterious roles in mammalian development and disease, therefore it is crucial to understand how these molecules are generated, sensed, and detoxified. The question of how oxidative stress connects to the epigenome, in particular, is important yet incompletely understood. Here we show that an epigenetic regulator, the methyl-CpG-binding protein ZBTB38, limits the basal cellular production of ROS, is induced by ROS, and is required to mount a proper response to oxidative stress. Molecularly, these functions depend on a deubiquitinase, USP9X, which interacts with ZBTB38, deubiquitinates it, and stabilizes it. We find that USP9X is itself stabilized by oxidative stress, and is required together with ZBTB38 to limit the basal generation of ROS, as well as the toxicity of an acute oxidative stress. Our data uncover a new nuclear target of USP9X, show that the USP9X/ZBTB38 axis limits, senses and detoxifies ROS, and provide a molecular link between oxidative stress and the epigenome.
Asunto(s)
Estrés Oxidativo , Proteínas Represoras/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Línea Celular Tumoral , Núcleo Celular/enzimología , Núcleo Celular/metabolismo , Humanos , Estabilidad Proteica , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Targeting the activation function-1 (AF-1) domain located in the N-terminus of the androgen receptor (AR) is an attractive therapeutic alternative to the current approaches to inhibit AR action in prostate cancer (PCa). Here we show that the AR AF-1 is bound by the cochaperone Bag-1L. Mutations in the AR interaction domain or loss of Bag-1L abrogate AR signaling and reduce PCa growth. Clinically, Bag-1L protein levels increase with progression to castration-resistant PCa (CRPC) and high levels of Bag-1L in primary PCa associate with a reduced clinical benefit from abiraterone when these tumors progress. Intriguingly, residues in Bag-1L important for its interaction with the AR AF-1 are within a potentially druggable pocket, implicating Bag-1L as a potential therapeutic target in PCa.
Asunto(s)
Antagonistas de Receptores Androgénicos/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Neoplasias de la Próstata/patología , Receptores Androgénicos/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Humanos , Masculino , Neoplasias de la Próstata/terapia , Unión Proteica , Mapas de Interacción de ProteínasRESUMEN
To maintain cellular homeostasis, subcellular organelles communicate with each other and form physical and functional networks through membrane contact sites coupled by protein tethers. In particular, endoplasmic reticulum (ER)-mitochondrial contacts (EMC) regulate diverse cellular activities such as metabolite exchange (Ca2+ and lipids), intracellular signaling, apoptosis, and autophagy. The significance of EMCs has been highlighted by reports indicating that EMC dysregulation is linked to neurodegenerative diseases. Therefore, obtaining a better understanding of the physical and functional components of EMCs should provide new insights into the pathogenesis of several neurodegenerative diseases. Here, we applied engineered ascorbate peroxidase (APEX) to map the proteome at EMCs in live HEK293 cells. APEX was targeted to the outer mitochondrial membrane, and proximity-labeled proteins were analyzed by stable isotope labeling with amino acids in culture (SILAC)-LC/MS-MS. We further refined the specificity of the proteins identified by combining biochemical subcellular fractionation to the protein isolation method. We identified 405 proteins with a 2.0-fold cutoff ratio (log base 2) in SILAC quantification from replicate experiments. We performed validation screening with a Split-Rluc8 complementation assay that identified reticulon 1A (RTN1A), an ER-shaping protein localized to EMCs as an EMC promoter. Proximity mapping augmented with biochemical fractionation and additional validation methods reported here could be useful to discover other components of EMCs, identify mitochondrial contacts with other organelles, and further unravel their communication.
Asunto(s)
Retículo Endoplásmico/metabolismo , Mitocondrias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Mapeo de Interacción de Proteínas/métodos , Ascorbato Peroxidasas/metabolismo , Prueba de Complementación Genética , Células HEK293 , Humanos , Indicadores y Reactivos/metabolismo , Marcaje Isotópico , Luciferasas de Renilla/genética , Luciferasas de Renilla/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/aislamiento & purificación , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Proyectos Piloto , Ingeniería de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
DNA methylation is an essential epigenetic mark in mammals that has to be re-established after each round of DNA replication. The protein UHRF1 is essential for this process; it has been proposed that the protein targets newly replicated DNA by cooperatively binding hemi-methylated DNA and H3K9me2/3, but this model leaves a number of questions unanswered. Here, we present evidence for a direct recruitment of UHRF1 by the replication machinery via DNA ligase 1 (LIG1). A histone H3K9-like mimic within LIG1 is methylated by G9a and GLP and, compared with H3K9me2/3, more avidly binds UHRF1. Interaction with methylated LIG1 promotes the recruitment of UHRF1 to DNA replication sites and is required for DNA methylation maintenance. These results further elucidate the function of UHRF1, identify a non-histone target of G9a and GLP, and provide an example of a histone mimic that coordinates DNA replication and DNA methylation maintenance.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , ADN Ligasa (ATP)/metabolismo , Metilación de ADN , Replicación del ADN , ADN/biosíntesis , Epigénesis Genética , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Proteínas Potenciadoras de Unión a CCAAT/química , Proteínas Potenciadoras de Unión a CCAAT/genética , ADN/genética , ADN Ligasa (ATP)/química , ADN Ligasa (ATP)/genética , Células Madre Embrionarias/enzimología , Células HEK293 , Células HeLa , Antígenos de Histocompatibilidad/química , Antígenos de Histocompatibilidad/genética , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Humanos , Lisina , Metilación , Ratones , Modelos Moleculares , Imitación Molecular , Mutación , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Transfección , Dominio Tudor , Ubiquitina-Proteína LigasasRESUMEN
The p110ß isoform of PI3K is preferentially activated in many tumors deficient in the phosphatase and tensin homolog (PTEN). However, the mechanism(s) linking PTEN loss to p110ß activation remain(s) mysterious. Here, we identify CRKL as a member of the class of PI3Kß-interacting proteins. Silencing CRKL expression in PTEN-null human cancer cells leads to a decrease in p110ß-dependent PI3K signaling and cell proliferation. In contrast, CRKL depletion does not impair p110α-mediated signaling. Further study showed that CRKL binds to tyrosine-phosphorylated p130Cas in PTEN-null cancer cells. Since Src family kinases are known both to be regulated by PTEN and to phosphorylate and activate p130Cas, we tested and found that Src inhibition cooperated with p110ß inhibition to suppress the growth of PTEN-null cells. These data suggest both a potential mechanism linking PTEN loss to p110ß activation and the possible benefit of dual inhibition of Src and PI3K for PTEN-null tumors.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Fosfohidrolasa PTEN/deficiencia , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase Ia/genética , Humanos , Neoplasias/genética , Proteínas Nucleares/genéticaRESUMEN
The continued evolution of modern mass spectrometry instrumentation and associated methods represents a critical component in efforts to decipher the molecular mechanisms which underlie normal physiology and understand how dysregulation of biological pathways contributes to human disease. The increasing scale of these experiments combined with the technological diversity of mass spectrometers presents several challenges for community-wide data access, analysis, and distribution. Here we detail a redesigned version of multiplierz, our Python software library which leverages our common application programming interface (mzAPI) for analysis and distribution of proteomic data. New features include support for a wider range of native mass spectrometry file types, interfaces to additional database search engines, compatibility with new reporting formats, and high-level tools to perform post-search proteomic analyses. A GUI desktop environment, mzDesktop, provides access to multiplierz functionality through a user friendly interface. multiplierz is available for download from: https://github.com/BlaisProteomics/multiplierz; and mzDesktop is available for download from: https://sourceforge.net/projects/multiplierz/.
Asunto(s)
Biología Computacional/métodos , Espectrometría de Masas/instrumentación , Proteómica/métodos , Programas Informáticos , Sistemas de Administración de Bases de Datos , Bases de Datos Factuales , Ecosistema , Humanos , Proteoma/análisis , Interfaz Usuario-ComputadorRESUMEN
Through an shRNA screen, we identified the protein arginine methyltransferase Prmt1 as a vulnerable intervention point in murine p53/Rb-null osteosarcomas, the human counterpart of which lacks effective therapeutic options. Depletion of Prmt1 in p53-deficient cells impaired tumor initiation and maintenance in vitro and in vivo Mechanistic studies reveal that translation-associated pathways were enriched for Prmt1 downstream targets, implicating Prmt1 in translation control. In particular, loss of Prmt1 led to a decrease in arginine methylation of the translation initiation complex, thereby disrupting its assembly and inhibiting translation. p53/Rb-null cells were sensitive to p53-induced translation stress, and analysis of human cancer cell line data from Project Achilles further revealed that Prmt1 and translation-associated pathways converged on the same functional networks. We propose that targeted therapy against Prmt1 and its associated translation-related pathways offer a mechanistic rationale for treatment of osteosarcomas and other cancers that exhibit dependencies on translation stress response. Cancer Res; 77(17); 4613-25. ©2017 AACR.
Asunto(s)
Neoplasias Óseas/patología , Osteosarcoma/patología , Biosíntesis de Proteínas , Proteína-Arginina N-Metiltransferasas/fisiología , Proteína de Retinoblastoma/fisiología , Proteína p53 Supresora de Tumor/fisiología , Animales , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Ratones , Ratones Noqueados , Osteosarcoma/genética , Osteosarcoma/metabolismo , Proteómica , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Serine/threonine kinase 40 (STK40) was originally identified as a distant homolog of Tribbles-family proteins. Despite accumulating data attesting to the importance of STK40 in a variety of different physiologic processes, little is known about its biological activity or mechanism of action. Here, we show that STK40 interacts with Constitutive Photomorphogenic Protein 1 (COP1), relying primarily on a C-terminal sequence analogous to the motif found in Tribbles proteins. In order to further elucidate structure-function relationships in STK40, we determined the crystal structure of the STK40 kinase homology domain at 2.5 Å resolution. The structure, together with ATP-binding assay results, show that STK40 is a pseudokinase, in which substitutions of conserved residues within the kinase domain prevent ATP binding. Although the structure of the kinase homology domain diverges from the analogous region of Trib1, the results reported here suggest functional parallels between STK40 and Tribbles-family proteins as COP1 adaptors.
Asunto(s)
Adenosina Trifosfato/química , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , Expresión Génica , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
COP1 proteins are E3 ubiquitin ligases that regulate phototropism in plants and target transcription factors for degradation in mammals. The substrate-binding region of COP1 resides within a WD40-repeat domain that also binds to Trib proteins, which are adaptors for C/EBPα degradation. Here we report structures of the human COP1 WD40 domain in isolation, and complexes of the human and Arabidopsis thaliana COP1 WD40 domains with the binding motif of Trib1. The human and Arabidopsis WD40 domains are seven-bladed ß propellers with an inserted loop on the bottom face of the first blade. The Trib1 peptide binds in an extended conformation to a highly conserved surface on the top face of the ß propeller, indicating a general mode for recognition of peptide motifs by COP1. Together, these studies identify the structural basis and key interactions for motif recognition by COP1, and hint at how Trib1 autoinhibition is overcome to target C/EBPα for degradation.