RESUMEN
IGHMBP2 is a nonessential, superfamily 1 DNA/RNA helicase that is mutated in patients with rare neuromuscular diseases SMARD1 and CMT2S. IGHMBP2 is implicated in translational and transcriptional regulation via biochemical association with ribosomal proteins, pre-rRNA processing factors, and tRNA-related species. To uncover the cellular consequences of perturbing IGHMBP2, we generated full and partial IGHMBP2 deletion K562 cell lines. Using polysome profiling and a nascent protein synthesis assay, we found that IGHMBP2 deletion modestly reduces global translation. We performed Ribo-seq and RNA-seq and identified diverse gene expression changes due to IGHMBP2 deletion, including ATF4 up-regulation. With recent studies showing the integrated stress response (ISR) can contribute to tRNA metabolism-linked neuropathies, we asked whether perturbing IGHMBP2 promotes ISR activation. We generated ATF4 reporter cell lines and found IGHMBP2 knockout cells demonstrate basal, chronic ISR activation. Our work expands upon the impact of IGHMBP2 in translation and elucidates molecular mechanisms that may link mutant IGHMBP2 to severe clinical phenotypes.
Asunto(s)
Proteínas de Unión al ADN , Biosíntesis de Proteínas , Estrés Fisiológico , Factores de Transcripción , Humanos , Biosíntesis de Proteínas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Estrés Fisiológico/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células K562 , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica , ARN de Transferencia/genética , ARN de Transferencia/metabolismoRESUMEN
Stress granules (SGs) are macromolecular assemblies that form under cellular stress. Formation of these condensates is driven by the condensation of RNA and RNA-binding proteins such as G3BPs. G3BPs condense into SGs following stress-induced translational arrest. Three G3BP paralogs (G3BP1, G3BP2A, and G3BP2B) have been identified in vertebrates. However, the contribution of different G3BP paralogs to stress granule formation and stress-induced gene expression changes is incompletely understood. Here, we identified key residues for G3BP condensation such as V11. This conserved amino acid is required for formation of the G3BP-Caprin-1 complex, hence promoting SG assembly. Total RNA sequencing and ribosome profiling revealed that disruption of G3BP condensation corresponds to changes in mRNA levels and ribosome engagement during the integrated stress response (ISR). Moreover, we found that G3BP2B preferentially condenses and promotes changes in mRNA expression under endoplasmic reticulum (ER) stress. Together, this work suggests that stress granule assembly promotes changes in gene expression under cellular stress, which is differentially regulated by G3BP paralogs.
RESUMEN
IGHMBP2 is a non-essential, superfamily 1 DNA/RNA helicase that is mutated in patients with rare neuromuscular diseases SMARD1 and CMT2S. IGHMBP2 is implicated in translational and transcriptional regulation via biochemical association with ribosomal proteins, pre-rRNA processing factors, and tRNA-related species. To uncover the cellular consequences of perturbing IGHMBP2, we generated full and partial IGHMBP2 deletion K562 cell lines. Using polysome profiling and a nascent protein synthesis assay, we found that IGHMBP2 deletion modestly reduces global translation. We performed Ribo-seq and RNA-seq and identified diverse gene expression changes due to IGHMBP2 deletion, including ATF4 upregulation. With recent studies showing the ISR can contribute to tRNA metabolism-linked neuropathies, we asked whether perturbing IGHMBP2 promotes ISR activation. We generated ATF4 reporter cell lines and found IGHMBP2 knockout cells demonstrate basal, chronic ISR activation. Our work expands upon the impact of IGHMBP2 in translation and elucidates molecular mechanisms that may link mutant IGHMBP2 to severe clinical phenotypes.
RESUMEN
Kinases play central roles in signaling cascades, relaying information from the outside to the inside of mammalian cells. De novo designed protein switches capable of interfacing with tyrosine kinase signaling pathways would open new avenues for controlling cellular behavior, but, so far, no such systems have been described. Here we describe the de novo design of two classes of protein switch that link phosphorylation by tyrosine and serine kinases to protein-protein association. In the first class, protein-protein association is required for phosphorylation by the kinase, while in the second class, kinase activity drives protein-protein association. We design systems that couple protein binding to kinase activity on the immunoreceptor tyrosine-based activation motif central to T-cell signaling, and kinase activity to reconstitution of green fluorescent protein fluorescence from fragments and the inhibition of the protease calpain. The designed switches are reversible and function in vitro and in cells with up to 40-fold activation of switching by phosphorylation.
Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Secuencias de Aminoácidos , Unión Competitiva , Proteínas de Unión al Calcio/farmacología , Calpaína/antagonistas & inhibidores , Calpaína/metabolismo , Catálisis , Dominio Catalítico , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Diseño de Fármacos , Genes Sintéticos , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Fosforilación , Fosfotirosina/metabolismo , Unión Proteica , Conformación Proteica , Dominios Proteicos , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/química , Proteínas Tirosina Quinasas/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Relación Estructura-Actividad , Familia-src Quinasas/metabolismoRESUMEN
Most glioblastomas (GBMs) achieve cellular immortality by acquiring a mutation in the telomerase reverse transcriptase (TERT) promoter. TERT promoter mutations create a binding site for a GA binding protein (GABP) transcription factor complex, whose assembly at the promoter is associated with TERT reactivation and telomere maintenance. Here, we demonstrate increased binding of a specific GABPB1L-isoform-containing complex to the mutant TERT promoter. Furthermore, we find that TERT promoter mutant GBM cells, unlike wild-type cells, exhibit a critical near-term dependence on GABPB1L for proliferation, notably also posttumor establishment in vivo. Up-regulation of the protein paralogue GABPB2, which is normally expressed at very low levels, can rescue this dependence. More importantly, when combined with frontline temozolomide (TMZ) chemotherapy, inducible GABPB1L knockdown and the associated TERT reduction led to an impaired DNA damage response that resulted in profoundly reduced growth of intracranial GBM tumors. Together, these findings provide insights into the mechanism of cancer-specific TERT regulation, uncover rapid effects of GABPB1L-mediated TERT suppression in GBM maintenance, and establish GABPB1L inhibition in combination with chemotherapy as a therapeutic strategy for TERT promoter mutant GBM.
Asunto(s)
Neoplasias Encefálicas/genética , Factor de Transcripción de la Proteína de Unión a GA/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Telomerasa/genética , Animales , Antineoplásicos Alquilantes/farmacología , Antineoplásicos Alquilantes/uso terapéutico , Astrocitos , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Proliferación Celular/genética , Daño del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Factor de Transcripción de la Proteína de Unión a GA/genética , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Células HEK293 , Humanos , Ratones , Mutación , Regiones Promotoras Genéticas/genética , Isoformas de Proteínas/metabolismo , Temozolomida/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Stochastic fluctuations at the transcriptional level contribute to isogenic cell-to-cell heterogeneity in mammalian cell populations. However, we still have no clear understanding of the repercussions of this heterogeneity, given the lack of tools to independently control mean expression and variability of a gene. Here, we engineer a synthetic circuit to modulate mean expression and heterogeneity of transgenes and endogenous human genes. The circuit, a Tunable Noise Rheostat (TuNR), consists of a transcriptional cascade of two inducible transcriptional activators, where the output mean and variance can be modulated by two orthogonal small molecule inputs. In this fashion, different combinations of the inputs can achieve the same mean but with different population variability. With TuNR, we achieve low basal expression, over 1000-fold expression of a transgene product, and up to 7-fold induction of the endogenous gene NGFR. Importantly, for the same mean expression level, we are able to establish varying degrees of heterogeneity in expression within an isogenic population, thereby decoupling gene expression noise from its mean. TuNR is therefore a modular tool that can be used in mammalian cells to enable direct interrogation of the implications of cell-to-cell variability.
Asunto(s)
Regulación de la Expresión Génica , Ácido Abscísico/farmacología , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Transactivadores/metabolismo , TransgenesRESUMEN
Natural products serve as chemical blueprints for most antibiotics in clinical use. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance mechanisms that shorten the clinical lifetime of any given class of antibiotics1. Virginiamycin acetyltransferase (Vat) enzymes are resistance proteins that provide protection against streptogramins2, potent antibiotics against Gram-positive bacteria that inhibit the bacterial ribosome3. Owing to the challenge of selectively modifying the chemically complex, 23-membered macrocyclic scaffold of group A streptogramins, analogues that overcome the resistance conferred by Vat enzymes have not been previously developed2. Here we report the design, synthesis, and antibacterial evaluation of group A streptogramin antibiotics with extensive structural variability. Using cryo-electron microscopy and forcefield-based refinement, we characterize the binding of eight analogues to the bacterial ribosome at high resolution, revealing binding interactions that extend into the peptidyl tRNA-binding site and towards synergistic binders that occupy the nascent peptide exit tunnel. One of these analogues has excellent activity against several streptogramin-resistant strains of Staphylococcus aureus, exhibits decreased rates of acetylation in vitro, and is effective at lowering bacterial load in a mouse model of infection. Our results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms.