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1.
EMBO J ; 43(7): 1164-1186, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38396301

RESUMEN

Ferroptosis is a regulated form of necrotic cell death caused by iron-dependent accumulation of oxidized phospholipids in cellular membranes, culminating in plasma membrane rupture (PMR) and cell lysis. PMR is also a hallmark of other types of programmed necrosis, such as pyroptosis and necroptosis, where it is initiated by dedicated pore-forming cell death-executing factors. However, whether ferroptosis-associated PMR is also actively executed by proteins or driven by osmotic pressure remains unknown. Here, we investigate a potential ferroptosis role of ninjurin-1 (NINJ1), a recently identified executor of pyroptosis-associated PMR. We report that NINJ1 oligomerizes during ferroptosis, and that Ninj1-deficiency protects macrophages and fibroblasts from ferroptosis-associated PMR. Mechanistically, we find that NINJ1 is dispensable for the initial steps of ferroptosis, such as lipid peroxidation, channel-mediated calcium influx, and cell swelling. In contrast, NINJ1 is required for early loss of plasma membrane integrity, which precedes complete PMR. Furthermore, NINJ1 mediates the release of cytosolic proteins and danger-associated molecular pattern (DAMP) molecules from ferroptotic cells, suggesting that targeting NINJ1 could be a therapeutic option to reduce ferroptosis-associated inflammation.


Asunto(s)
Alarminas , Ferroptosis , Humanos , Necrosis/metabolismo , Muerte Celular , Membrana Celular/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo
2.
Nature ; 618(7967): 1065-1071, 2023 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-37198476

RESUMEN

Eukaryotic cells can undergo different forms of programmed cell death, many of which culminate in plasma membrane rupture as the defining terminal event1-7. Plasma membrane rupture was long thought to be driven by osmotic pressure, but it has recently been shown to be in many cases an active process, mediated by the protein ninjurin-18 (NINJ1). Here we resolve the structure of NINJ1 and the mechanism by which it ruptures membranes. Super-resolution microscopy reveals that NINJ1 clusters into structurally diverse assemblies in the membranes of dying cells, in particular large, filamentous assemblies with branched morphology. A cryo-electron microscopy structure of NINJ1 filaments shows a tightly packed fence-like array of transmembrane α-helices. Filament directionality and stability is defined by two amphipathic α-helices that interlink adjacent filament subunits. The NINJ1 filament features a hydrophilic side and a hydrophobic side, and molecular dynamics simulations show that it can stably cap membrane edges. The function of the resulting supramolecular arrangement was validated by site-directed mutagenesis. Our data thus suggest that, during lytic cell death, the extracellular α-helices of NINJ1 insert into the plasma membrane to polymerize NINJ1 monomers into amphipathic filaments that rupture the plasma membrane. The membrane protein NINJ1 is therefore an interactive component of the eukaryotic cell membrane that functions as an in-built breaking point in response to activation of cell death.


Asunto(s)
Moléculas de Adhesión Celular Neuronal , Muerte Celular , Membrana Celular , Factores de Crecimiento Nervioso , Animales , Humanos , Ratones , Moléculas de Adhesión Celular Neuronal/química , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Moléculas de Adhesión Celular Neuronal/ultraestructura , Membrana Celular/metabolismo , Membrana Celular/patología , Membrana Celular/ultraestructura , Microscopía por Crioelectrón , Factores de Crecimiento Nervioso/química , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/ultraestructura , Mutagénesis Sitio-Dirigida , Biopolímeros/química , Biopolímeros/genética , Biopolímeros/metabolismo
3.
Nucleic Acids Res ; 51(1): 182-197, 2023 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-36537232

RESUMEN

Alkaline exonucleases (AE) are present in several large DNA viruses including bacteriophage λ and herpesviruses, where they play roles in viral DNA processing during genome replication. Given the genetic conservation of AEs across viruses infecting different kingdoms of life, these enzymes likely assume central roles in the lifecycles of viruses where they have yet to be well characterized. Here, we applied a structure-guided functional analysis of the bifunctional AE in the oncogenic human gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV), called SOX. In addition to identifying a preferred DNA substrate preference for SOX, we define key residues important for DNA binding and DNA processing, and how SOX activity on DNA partially overlaps with its functionally separable cleavage of mRNA. By engineering these SOX mutants into KSHV, we reveal roles for its DNase activity in viral gene expression and infectious virion production. Our results provide mechanistic insight into gammaherpesviral AE activity as well as areas of functional conservation between this mammalian virus AE and its distant relative in phage λ.


Asunto(s)
Exonucleasas , Herpesvirus Humano 8 , Animales , Humanos , ADN Viral/metabolismo , Exonucleasas/genética , Expresión Génica , Regulación Viral de la Expresión Génica , Herpesvirus Humano 8/metabolismo , Mamíferos/genética , Virión/metabolismo , Replicación Viral
5.
Cell Rep ; 37(3): 109841, 2021 10 19.
Artículo en Inglés | MEDLINE | ID: mdl-34624207

RESUMEN

Nonstructural protein 1 (nsp1) is a coronavirus (CoV) virulence factor that restricts cellular gene expression by inhibiting translation through blocking the mRNA entry channel of the 40S ribosomal subunit and by promoting mRNA degradation. We perform a detailed structure-guided mutational analysis of severe acute respiratory syndrome (SARS)-CoV-2 nsp1, revealing insights into how it coordinates these activities against host but not viral mRNA. We find that residues in the N-terminal and central regions of nsp1 not involved in docking into the 40S mRNA entry channel nonetheless stabilize its association with the ribosome and mRNA, both enhancing its restriction of host gene expression and enabling mRNA containing the SARS-CoV-2 leader sequence to escape translational repression. These data support a model in which viral mRNA binding functionally alters the association of nsp1 with the ribosome, which has implications for drug targeting and understanding how engineered or emerging mutations in SARS-CoV-2 nsp1 could attenuate the virus.


Asunto(s)
COVID-19/genética , Regulación Viral de la Expresión Génica , SARS-CoV-2/genética , Proteínas no Estructurales Virales/metabolismo , Anisotropía , COVID-19/inmunología , Análisis Mutacional de ADN , Femenino , Perfilación de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Cinética , Mutación , Fenotipo , Mutación Puntual , Biosíntesis de Proteínas , Dominios Proteicos , Estabilidad del ARN , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Ribosomas/metabolismo
6.
Elife ; 102021 06 04.
Artículo en Inglés | MEDLINE | ID: mdl-34085923

RESUMEN

RNA abundance is generally sensitive to perturbations in decay and synthesis rates, but crosstalk between RNA polymerase II transcription and cytoplasmic mRNA degradation often leads to compensatory changes in gene expression. Here, we reveal that widespread mRNA decay during early apoptosis represses RNAPII transcription, indicative of positive (rather than compensatory) feedback. This repression requires active cytoplasmic mRNA degradation, which leads to impaired recruitment of components of the transcription preinitiation complex to promoter DNA. Importin α/ß-mediated nuclear import is critical for this feedback signaling, suggesting that proteins translocating between the cytoplasm and nucleus connect mRNA decay to transcription. We also show that an analogous pathway activated by viral nucleases similarly depends on nuclear protein import. Collectively, these data demonstrate that accelerated mRNA decay leads to the repression of mRNA transcription, thereby amplifying the shutdown of gene expression. This highlights a conserved gene regulatory mechanism by which cells respond to threats.


Asunto(s)
Apoptosis , Neoplasias/metabolismo , ARN Polimerasa II/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , ARN Neoplásico/metabolismo , Transcripción Genética , Transporte Activo de Núcleo Celular , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Citoplasma/genética , Citoplasma/metabolismo , Retroalimentación Fisiológica , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , ARN Polimerasa II/genética , ARN Mensajero/genética , ARN Neoplásico/genética , Factores de Tiempo , alfa Carioferinas/metabolismo , beta Carioferinas/metabolismo
7.
J Biol Chem ; 295(37): 12910-12934, 2020 09 11.
Artículo en Inglés | MEDLINE | ID: mdl-32661197

RESUMEN

Few human pathogens have been the focus of as much concentrated worldwide attention as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of COVID-19. Its emergence into the human population and ensuing pandemic came on the heels of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), two other highly pathogenic coronavirus spillovers, which collectively have reshaped our view of a virus family previously associated primarily with the common cold. It has placed intense pressure on the collective scientific community to develop therapeutics and vaccines, whose engineering relies on a detailed understanding of coronavirus biology. Here, we present the molecular virology of coronavirus infection, including its entry into cells, its remarkably sophisticated gene expression and replication mechanisms, its extensive remodeling of the intracellular environment, and its multifaceted immune evasion strategies. We highlight aspects of the viral life cycle that may be amenable to antiviral targeting as well as key features of its biology that await discovery.


Asunto(s)
Infecciones por Coronavirus/virología , Coronavirus/fisiología , Regulación Viral de la Expresión Génica , Fenómenos Fisiológicos de los Virus , Animales , Antígenos Virales/inmunología , Coronavirus/genética , Coronavirus/inmunología , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/inmunología , Interacciones Huésped-Patógeno , Humanos
8.
PLoS Pathog ; 16(2): e1008269, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-32032393

RESUMEN

In mammalian cells, widespread acceleration of cytoplasmic mRNA degradation is linked to impaired RNA polymerase II (Pol II) transcription. This mRNA decay-induced transcriptional repression occurs during infection with gammaherpesviruses including Kaposi's sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68), which encode an mRNA endonuclease that initiates widespread RNA decay. Here, we show that MHV68-induced mRNA decay leads to a genome-wide reduction of Pol II occupancy at mammalian promoters. This reduced Pol II occupancy is accompanied by down-regulation of multiple Pol II subunits and TFIIB in the nucleus of infected cells, as revealed by mass spectrometry-based global measurements of protein abundance. Viral genes, despite the fact that they require Pol II for transcription, escape transcriptional repression. Protection is not governed by viral promoter sequences; instead, location on the viral genome is both necessary and sufficient to escape the transcriptional repression effects of mRNA decay. We propose a model in which the ability to escape from transcriptional repression is linked to the localization of viral DNA within replication compartments, providing a means for these viruses to counteract decay-induced transcript loss.


Asunto(s)
Infecciones por Herpesviridae/metabolismo , Herpesvirus Humano 8/fisiología , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Estabilidad del ARN , Rhadinovirus/fisiología , Replicación Viral , Animales , Endonucleasas/genética , Endonucleasas/metabolismo , Genoma Viral , Infecciones por Herpesviridae/genética , Ratones , Células 3T3 NIH , ARN Polimerasa II/genética , Factor de Transcripción TFIIB/genética , Factor de Transcripción TFIIB/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo
9.
Crit Rev Biochem Mol Biol ; 54(4): 385-398, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31656086

RESUMEN

Transcription and RNA decay are key determinants of gene expression; these processes are typically considered as the uncoupled beginning and end of the messenger RNA (mRNA) lifecycle. Here we describe the growing number of studies demonstrating interplay between these spatially disparate processes in eukaryotes. Specifically, cells can maintain mRNA levels by buffering against changes in mRNA stability or transcription, and can also respond to virally induced accelerated decay by reducing RNA polymerase II gene expression. In addition to these global responses, there is also evidence that mRNAs containing a premature stop codon can cause transcriptional upregulation of homologous genes in a targeted fashion. In each of these systems, RNA binding proteins (RBPs), particularly those involved in mRNA degradation, are critical for cytoplasmic to nuclear communication. Although their specific mechanistic contributions are yet to be fully elucidated, differential trafficking of RBPs between subcellular compartments are likely to play a central role in regulating this gene expression feedback pathway.


Asunto(s)
Citoplasma/genética , Estabilidad del ARN/genética , ARN Mensajero/genética , Transcripción Genética , Animales , Núcleo Celular/genética , Núcleo Celular/metabolismo , Codón de Terminación/genética , Citoplasma/metabolismo , Exorribonucleasas/metabolismo , Expresión Génica , Homeostasis/genética , Humanos , Infecciones/genética , Proteínas Asociadas a Microtúbulos/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética
10.
Elife ; 72018 10 03.
Artículo en Inglés | MEDLINE | ID: mdl-30281021

RESUMEN

Alterations in global mRNA decay broadly impact multiple stages of gene expression, although signals that connect these processes are incompletely defined. Here, we used tandem mass tag labeling coupled with mass spectrometry to reveal that changing the mRNA decay landscape, as frequently occurs during viral infection, results in subcellular redistribution of RNA binding proteins (RBPs) in human cells. Accelerating Xrn1-dependent mRNA decay through expression of a gammaherpesviral endonuclease drove nuclear translocation of many RBPs, including poly(A) tail-associated proteins. Conversely, cells lacking Xrn1 exhibited changes in the localization or abundance of numerous factors linked to mRNA turnover. Using these data, we uncovered a new role for relocalized cytoplasmic poly(A) binding protein in repressing recruitment of TATA binding protein and RNA polymerase II to promoters. Collectively, our results show that changes in cytoplasmic mRNA decay can directly impact protein localization, providing a mechanism to connect seemingly distal stages of gene expression.


Asunto(s)
Regulación de la Expresión Génica , Transporte de Proteínas , Estabilidad del ARN , Transporte de ARN , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Transcripción Genética , Exorribonucleasas/metabolismo , Células HEK293 , Humanos , Espectrometría de Masas , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , ARN Polimerasa II/metabolismo , Coloración y Etiquetado , Proteína de Unión a TATA-Box/metabolismo
11.
Nat Biotechnol ; 36(2): 179-189, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29251726

RESUMEN

Combinatorial genetic screening using CRISPR-Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks. However, current methods suffer from interference between the single-guide RNAs (sgRNAs) and from limited gene targeting activity. To increase the efficiency of combinatorial screening, we employ orthogonal Cas9 enzymes from Staphylococcus aureus and Streptococcus pyogenes. We used machine learning to establish S. aureus Cas9 sgRNA design rules and paired S. aureus Cas9 with S. pyogenes Cas9 to achieve dual targeting in a high fraction of cells. We also developed a lentiviral vector and cloning strategy to generate high-complexity pooled dual-knockout libraries to identify synthetic lethal and buffering gene pairs across multiple cell types, including MAPK pathway genes and apoptotic genes. Our orthologous approach also enabled a screen combining gene knockouts with transcriptional activation, which revealed genetic interactions with TP53. The "Big Papi" (paired aureus and pyogenes for interactions) approach described here will be widely applicable for the study of combinatorial phenotypes.


Asunto(s)
Sistemas CRISPR-Cas/genética , Epistasis Genética/genética , Pruebas Genéticas , ARN Guía de Kinetoplastida/genética , Apoptosis/genética , Técnicas de Inactivación de Genes , Marcación de Gen , Humanos , Aprendizaje Automático , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Transducción de Señal/genética , Staphylococcus aureus/genética , Streptococcus pyogenes/genética , Proteína p53 Supresora de Tumor/genética
12.
Science ; 358(6365): 888-893, 2017 11 17.
Artículo en Inglés | MEDLINE | ID: mdl-29146805

RESUMEN

Robust innate immune detection of rapidly evolving pathogens is critical for host defense. Nucleotide-binding domain leucine-rich repeat (NLR) proteins function as cytosolic innate immune sensors in plants and animals. However, the structural basis for ligand-induced NLR activation has so far remained unknown. NAIP5 (NLR family, apoptosis inhibitory protein 5) binds the bacterial protein flagellin and assembles with NLRC4 to form a multiprotein complex called an inflammasome. Here we report the cryo-electron microscopy structure of the assembled ~1.4-megadalton flagellin-NAIP5-NLRC4 inflammasome, revealing how a ligand activates an NLR. Six distinct NAIP5 domains contact multiple conserved regions of flagellin, prying NAIP5 into an open and active conformation. We show that innate immune recognition of multiple ligand surfaces is a generalizable strategy that limits pathogen evolution and immune escape.


Asunto(s)
Flagelina/inmunología , Interacciones Huésped-Patógeno/inmunología , Inflamasomas/inmunología , Proteína Inhibidora de la Apoptosis Neuronal/inmunología , Animales , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/inmunología , Proteínas Reguladoras de la Apoptosis/ultraestructura , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/inmunología , Proteínas de Unión al Calcio/ultraestructura , Microscopía por Crioelectrón , Flagelina/química , Flagelina/ultraestructura , Células HEK293 , Humanos , Inmunidad Innata , Inflamasomas/química , Inflamasomas/ultraestructura , Legionella pneumophila , Ratones , Mutación , Proteína Inhibidora de la Apoptosis Neuronal/química , Proteína Inhibidora de la Apoptosis Neuronal/genética , Dominios Proteicos
13.
Cell Syst ; 3(3): 302-316.e4, 2016 09 28.
Artículo en Inglés | MEDLINE | ID: mdl-27684187

RESUMEN

Genome-scale expression studies and comprehensive loss-of-function genetic screens have focused almost exclusively on the highest confidence candidate genes. Here, we describe a strategy for characterizing the lower confidence candidates identified by such approaches. We interrogated 177 genes that we classified as essential for the proliferation of cancer cells exhibiting constitutive ß-catenin activity and integrated data for each of the candidates, derived from orthogonal short hairpin RNA (shRNA) knockdown and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated gene editing knockout screens, to yield 69 validated genes. We then characterized the relationships between sets of these genes using complementary assays: medium-throughput stable isotope labeling by amino acids in cell culture (SILAC)-based mass spectrometry, yielding 3,639 protein-protein interactions, and a CRISPR-mediated pairwise double knockout screen, yielding 375 combinations exhibiting greater- or lesser-than-additive phenotypic effects indicating genetic interactions. These studies identify previously unreported regulators of ß-catenin, define functional networks required for the survival of ß-catenin-active cancers, and provide an experimental strategy that may be applied to define other signaling networks.


Asunto(s)
Proteómica , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica , Terapia Genética , Humanos , Neoplasias , ARN Guía de Kinetoplastida , ARN Interferente Pequeño , beta Catenina
14.
FEBS J ; 282(8): 1383-93, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25728500

RESUMEN

Functional genomics attempts to understand the genome by perturbing the flow of information from DNA to RNA to protein, in order to learn how gene dysfunction leads to disease. CRISPR/Cas9 technology is the newest tool in the geneticist's toolbox, allowing researchers to edit DNA with unprecedented ease, speed and accuracy, and representing a novel means to perform genome-wide genetic screens to discover gene function. In this review, we first summarize the discovery and characterization of CRISPR/Cas9, and then compare it to other genome engineering technologies. We discuss its initial use in screening applications, with a focus on optimizing on-target activity and minimizing off-target effects. Finally, we comment on future challenges and opportunities afforded by this technology.


Asunto(s)
Proteínas Asociadas a CRISPR/fisiología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Regulación de la Expresión Génica , Ingeniería Genética/métodos , Pruebas Genéticas , Genoma Humano , Edición de ARN/genética , Humanos
15.
Nat Biotechnol ; 32(12): 1262-7, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25184501

RESUMEN

Components of the prokaryotic clustered, regularly interspaced, short palindromic repeats (CRISPR) loci have recently been repurposed for use in mammalian cells. The CRISPR-associated (Cas)9 can be programmed with a single guide RNA (sgRNA) to generate site-specific DNA breaks, but there are few known rules governing on-target efficacy of this system. We created a pool of sgRNAs, tiling across all possible target sites of a panel of six endogenous mouse and three endogenous human genes and quantitatively assessed their ability to produce null alleles of their target gene by antibody staining and flow cytometry. We discovered sequence features that improved activity, including a further optimization of the protospacer-adjacent motif (PAM) of Streptococcus pyogenes Cas9. The results from 1,841 sgRNAs were used to construct a predictive model of sgRNA activity to improve sgRNA design for gene editing and genetic screens. We provide an online tool for the design of highly active sgRNAs for any gene of interest.


Asunto(s)
Sistemas CRISPR-Cas/genética , Silenciador del Gen , Marcación de Gen , ARN/genética , Animales , Citometría de Flujo , Humanos , Ratones , Streptococcus pyogenes/genética
16.
Science ; 343(6166): 84-87, 2014 Jan 03.
Artículo en Inglés | MEDLINE | ID: mdl-24336571

RESUMEN

The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.


Asunto(s)
Supervivencia Celular/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Resistencia a Antineoplásicos/genética , Pruebas Genéticas/métodos , Melanoma/genética , Células Madre Pluripotentes/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Cullin/genética , Técnicas de Inactivación de Genes , Biblioteca de Genes , Genes de Neurofibromatosis 1 , Genes de la Neurofibromatosis 2 , Sitios Genéticos , Estudio de Asociación del Genoma Completo , Humanos , Indoles/uso terapéutico , Lentivirus , Complejo Mediador/genética , Melanoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Selección Genética , Sulfonamidas/uso terapéutico , Factores de Transcripción/genética , Vemurafenib , Quinasas raf/antagonistas & inhibidores
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