RESUMEN
Mutations causing amyotrophic lateral sclerosis (ALS) often affect the condensation properties of RNA-binding proteins (RBPs). However, the role of RBP condensation in the specificity and function of protein-RNA complexes remains unclear. We created a series of TDP-43 C-terminal domain (CTD) variants that exhibited a gradient of low to high condensation propensity, as observed in vitro and by nuclear mobility and foci formation. Notably, a capacity for condensation was required for efficient TDP-43 assembly on subsets of RNA-binding regions, which contain unusually long clusters of motifs of characteristic types and density. These "binding-region condensates" are promoted by homomeric CTD-driven interactions and required for efficient regulation of a subset of bound transcripts, including autoregulation of TDP-43 mRNA. We establish that RBP condensation can occur in a binding-region-specific manner to selectively modulate transcriptome-wide RNA regulation, which has implications for remodeling RNA networks in the context of signaling, disease, and evolution.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Regiones no Traducidas 3'/genética , Secuencia de Bases , Núcleo Celular/metabolismo , Células HEK293 , Células HeLa , Homeostasis , Humanos , Mutación/genética , Motivos de Nucleótidos/genética , Transición de Fase , Mutación Puntual/genética , Poli A/metabolismo , Unión Proteica , Multimerización de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Eliminación de SecuenciaRESUMEN
Understanding the functional consequences of single-nucleotide variants is critical to uncovering the genetic underpinnings of diseases, but technologies to characterize variants are limiting. Here, we leverage CRISPR-Cas9 cytosine base editors in pooled screens to scalably assay variants at endogenous loci in mammalian cells. We benchmark the performance of base editors in positive and negative selection screens, identifying known loss-of-function mutations in BRCA1 and BRCA2 with high precision. To demonstrate the utility of base editor screens to probe small molecule-protein interactions, we screen against BH3 mimetics and PARP inhibitors, identifying point mutations that confer drug sensitivity or resistance. We also create a library of single guide RNAs (sgRNAs) predicted to generate 52,034 ClinVar variants in 3,584 genes and conduct screens in the presence of cellular stressors, identifying loss-of-function variants in numerous DNA damage repair genes. We anticipate that this screening approach will be broadly useful to readily and scalably functionalize genetic variants.
Asunto(s)
Edición Génica , Variación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Alelos , Proteína BRCA1/genética , Proteína BRCA2/genética , Secuencia de Bases , Dominio Catalítico , Línea Celular Tumoral , Humanos , Mutación con Pérdida de Función , Mutagénesis/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Mutación Puntual/genética , Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/genética , Reproducibilidad de los Resultados , Selección Genética , Proteína bcl-X/genéticaRESUMEN
Although base editors are widely used to install targeted point mutations, the factors that determine base editing outcomes are not well understood. We characterized sequence-activity relationships of 11 cytosine and adenine base editors (CBEs and ABEs) on 38,538 genomically integrated targets in mammalian cells and used the resulting outcomes to train BE-Hive, a machine learning model that accurately predicts base editing genotypic outcomes (R ≈ 0.9) and efficiency (R ≈ 0.7). We corrected 3,388 disease-associated SNVs with ≥90% precision, including 675 alleles with bystander nucleotides that BE-Hive correctly predicted would not be edited. We discovered determinants of previously unpredictable C-to-G, or C-to-A editing and used these discoveries to correct coding sequences of 174 pathogenic transversion SNVs with ≥90% precision. Finally, we used insights from BE-Hive to engineer novel CBE variants that modulate editing outcomes. These discoveries illuminate base editing, enable editing at previously intractable targets, and provide new base editors with improved editing capabilities.
Asunto(s)
Edición Génica/métodos , Aprendizaje Automático , Animales , Biblioteca de Genes , Humanos , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Mutación Puntual , ARN Guía de Kinetoplastida/metabolismoRESUMEN
Sickle cell disease (SCD) is caused by a point mutation in the ß-globin gene that creates hemoglobin S (HbS). Upon deoxygenation, HbS forms long polymers that distort the shape of red blood cells, causing hemolysis and vaso-occlusion. Voxelotor inhibits HbS polymerization, the root cause of SCD complications. To view this Bench to Bedside, open or download the PDF.
Asunto(s)
Anemia de Células Falciformes/genética , Benzaldehídos/uso terapéutico , Hemoglobina Falciforme/antagonistas & inhibidores , Pirazinas/uso terapéutico , Pirazoles/uso terapéutico , Globinas beta/genética , Anemia de Células Falciformes/epidemiología , Hemoglobina Falciforme/genética , Humanos , Mutación Puntual/genética , Polimerizacion/efectos de los fármacosRESUMEN
To discover regulatory elements driving the specificity of gene expression in different cell types and regions of the developing human brain, we generated an atlas of open chromatin from nine dissected regions of the mid-gestation human telencephalon, as well as microdissected upper and deep layers of the prefrontal cortex. We identified a subset of open chromatin regions (OCRs), termed predicted regulatory elements (pREs), that are likely to function as developmental brain enhancers. pREs showed temporal, regional, and laminar differences in chromatin accessibility and were correlated with gene expression differences across regions and gestational ages. We identified two functional de novo variants in a pRE for autism risk gene SLC6A1, and using CRISPRa, demonstrated that this pRE regulates SCL6A1. Additionally, mouse transgenic experiments validated enhancer activity for pREs proximal to FEZF2 and BCL11A. Thus, this atlas serves as a resource for decoding neurodevelopmental gene regulation in health and disease.
Asunto(s)
Cromatina/genética , Cromatina/metabolismo , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica/genética , Corteza Prefrontal/embriología , Telencéfalo/embriología , Animales , Trastorno Autístico/genética , Línea Celular , Secuenciación de Inmunoprecipitación de Cromatina , Eucromatina/genética , Proteínas Transportadoras de GABA en la Membrana Plasmática/genética , Ontología de Genes , Predisposición Genética a la Enfermedad , Edad Gestacional , Humanos , Ratones , Ratones Transgénicos , Motivos de Nucleótidos , Mutación Puntual , Corteza Prefrontal/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Análisis Espacio-Temporal , Telencéfalo/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Inflammatory bowel disease (IBD) is a chronic inflammatory disease associated with increased risk of gastrointestinal cancers. We whole-genome sequenced 446 colonic crypts from 46 IBD patients and compared these to 412 crypts from 41 non-IBD controls from our previous publication on the mutation landscape of the normal colon. The average mutation rate of affected colonic epithelial cells is 2.4-fold that of healthy colon, and this increase is mostly driven by acceleration of mutational processes ubiquitously observed in normal colon. In contrast to the normal colon, where clonal expansions outside the confines of the crypt are rare, we observed widespread millimeter-scale clonal expansions. We discovered non-synonymous mutations in ARID1A, FBXW7, PIGR, ZC3H12A, and genes in the interleukin 17 and Toll-like receptor pathways, under positive selection in IBD. These results suggest distinct selection mechanisms in the colitis-affected colon and that somatic mutations potentially play a causal role in IBD pathogenesis.
Asunto(s)
Evolución Clonal/genética , Colitis/genética , Enfermedades Inflamatorias del Intestino/genética , Tasa de Mutación , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/genética , Evolución Clonal/inmunología , Colitis/metabolismo , Colitis Ulcerosa/genética , Colitis Ulcerosa/metabolismo , Enfermedad de Crohn/genética , Enfermedad de Crohn/metabolismo , Proteínas de Unión al ADN/genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Femenino , Humanos , Mutación INDEL , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Interleucina-17/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Persona de Mediana Edad , Filogenia , Mutación Puntual , Receptores de Superficie Celular/genética , Ribonucleasas/genética , Receptores Toll-Like/genética , Factores de Transcripción/genética , Secuenciación Completa del GenomaRESUMEN
Cancer develops as a result of somatic mutation and clonal selection, but quantitative measures of selection in cancer evolution are lacking. We adapted methods from molecular evolution and applied them to 7,664 tumors across 29 cancer types. Unlike species evolution, positive selection outweighs negative selection during cancer development. On average, <1 coding base substitution/tumor is lost through negative selection, with purifying selection almost absent outside homozygous loss of essential genes. This allows exome-wide enumeration of all driver coding mutations, including outside known cancer genes. On average, tumors carry â¼4 coding substitutions under positive selection, ranging from <1/tumor in thyroid and testicular cancers to >10/tumor in endometrial and colorectal cancers. Half of driver substitutions occur in yet-to-be-discovered cancer genes. With increasing mutation burden, numbers of driver mutations increase, but not linearly. We systematically catalog cancer genes and show that genes vary extensively in what proportion of mutations are drivers versus passengers.
Asunto(s)
Neoplasias/genética , Neoplasias/patología , Humanos , Mutación INDEL , Inestabilidad de Microsatélites , Modelos Genéticos , Tasa de Mutación , Neoplasias/inmunología , Mutación Puntual , Polimorfismo de Nucleótido Simple , Selección GenéticaRESUMEN
The molecular basis of the incomplete penetrance of monogenic disorders is unclear. We describe here eight related individuals with autosomal recessive TIRAP deficiency. Life-threatening staphylococcal disease occurred during childhood in the proband, but not in the other seven homozygotes. Responses to all Toll-like receptor 1/2 (TLR1/2), TLR2/6, and TLR4 agonists were impaired in the fibroblasts and leukocytes of all TIRAP-deficient individuals. However, the whole-blood response to the TLR2/6 agonist staphylococcal lipoteichoic acid (LTA) was abolished only in the index case individual, the only family member lacking LTA-specific antibodies (Abs). This defective response was reversed in the patient, but not in interleukin-1 receptor-associated kinase 4 (IRAK-4)-deficient individuals, by anti-LTA monoclonal antibody (mAb). Anti-LTA mAb also rescued the macrophage response in mice lacking TIRAP, but not TLR2 or MyD88. Thus, acquired anti-LTA Abs rescue TLR2-dependent immunity to staphylococcal LTA in individuals with inherited TIRAP deficiency, accounting for incomplete penetrance. Combined TIRAP and anti-LTA Ab deficiencies underlie staphylococcal disease in this patient.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Lipopolisacáridos/metabolismo , Glicoproteínas de Membrana/deficiencia , Receptores de Interleucina-1/deficiencia , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/inmunología , Ácidos Teicoicos/metabolismo , Inmunidad Adaptativa , Niño , Femenino , Fibroblastos/metabolismo , Humanos , Inmunidad Innata , Lipopolisacáridos/inmunología , Macrófagos/inmunología , Masculino , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/genética , Monocitos/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Linaje , Fagocitos/metabolismo , Mutación Puntual , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , Receptores de Interleucina-1/análisis , Receptores de Interleucina-1/genética , Infecciones Estafilocócicas/tratamiento farmacológico , Ácidos Teicoicos/inmunología , Receptor Toll-Like 2/metabolismo , Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismoRESUMEN
Oncogenic activation of MYC in cancers predominantly involves increased transcription rather than coding region mutations. However, MYC-dependent lymphomas frequently acquire point mutations in the MYC phosphodegron, including at threonine 58 (T58), where phosphorylation permits binding via the FBW7 ubiquitin ligase triggering MYC degradation. To understand how T58 phosphorylation functions in normal cell physiology, we introduced an alanine mutation at T58 (T58A) into the endogenous c-Myc locus in the mouse germline. While MYC-T58A mice develop normally, lymphomas and myeloid leukemias emerge in â¼60% of adult homozygous T58A mice. We found that primitive hematopoietic progenitor cells from MYC-T58A mice exhibit aberrant self-renewal normally associated with hematopoietic stem cells (HSCs) and up-regulate a subset of MYC target genes important in maintaining stem/progenitor cell balance. In lymphocytes, genomic occupancy by MYC-T58A was increased at all promoters compared with WT MYC, while genes differentially expressed in a T58A-dependent manner were significantly more proximal to MYC-bound enhancers. MYC-T58A lymphocyte progenitors exhibited metabolic alterations and decreased activation of inflammatory and apoptotic pathways. Our data demonstrate that a single point mutation stabilizing MYC is sufficient to skew target gene expression, producing a profound gain of function in multipotential hematopoietic progenitors associated with self-renewal and initiation of lymphomas and leukemias.
Asunto(s)
Proteína 7 que Contiene Repeticiones F-Box-WD , Neoplasias Hematológicas , Linfoma , Proteínas Proto-Oncogénicas c-myc , Animales , Ratones , Células Germinativas/metabolismo , Células Madre Hematopoyéticas/metabolismo , Mutación Puntual , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismoRESUMEN
Comparative analyses have identified genomic regions potentially involved in human evolution but do not directly assess function. Human accelerated regions (HARs) represent conserved genomic loci with elevated divergence in humans. If some HARs regulate human-specific social and behavioral traits, then mutations would likely impact cognitive and social disorders. Strikingly, rare biallelic point mutations-identified by whole-genome and targeted "HAR-ome" sequencing-showed a significant excess in individuals with ASD whose parents share common ancestry compared to familial controls, suggesting a contribution in 5% of consanguineous ASD cases. Using chromatin interaction sequencing, massively parallel reporter assays (MPRA), and transgenic mice, we identified disease-linked, biallelic HAR mutations in active enhancers for CUX1, PTBP2, GPC4, CDKL5, and other genes implicated in neural function, ASD, or both. Our data provide genetic evidence that specific HARs are essential for normal development, consistent with suggestions that their evolutionary changes may have altered social and/or cognitive behavior. PAPERCLIP.
Asunto(s)
Trastorno del Espectro Autista/genética , Cognición , Predisposición Genética a la Enfermedad , Neurogénesis/genética , Mutación Puntual , Conducta Social , Alelos , Animales , Corteza Cerebral/metabolismo , Dosificación de Gen , Variación Genética , Genoma Humano , Proteínas de Homeodominio/genética , Humanos , Intrones , Ratones , Ratones Transgénicos , Proteínas Nucleares/genética , Sitios de Carácter Cuantitativo , Elementos Reguladores de la Transcripción , Proteínas Represoras/genética , Factores de TranscripciónRESUMEN
Cancer cells acquire pathological phenotypes through accumulation of mutations that perturb signaling networks. However, global analysis of these events is currently limited. Here, we identify six types of network-attacking mutations (NAMs), including changes in kinase and SH2 modulation, network rewiring, and the genesis and extinction of phosphorylation sites. We developed a computational platform (ReKINect) to identify NAMs and systematically interpreted the exomes and quantitative (phospho-)proteomes of five ovarian cancer cell lines and the global cancer genome repository. We identified and experimentally validated several NAMs, including PKCγ M501I and PKD1 D665N, which encode specificity switches analogous to the appearance of kinases de novo within the kinome. We discover mutant molecular logic gates, a drift toward phospho-threonine signaling, weakening of phosphorylation motifs, and kinase-inactivating hotspots in cancer. Our method pinpoints functional NAMs, scales with the complexity of cancer genomes and cell signaling, and may enhance our capability to therapeutically target tumor-specific networks.
Asunto(s)
Neoplasias Ováricas/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Transducción de Señal , Femenino , Humanos , Almacenamiento y Recuperación de la Información , Modelos Moleculares , Mutación Puntual , Proteínas Quinasas/química , Programas InformáticosRESUMEN
Most cell-surface receptors for cytokines and growth factors signal as dimers, but it is unclear whether remodeling receptor dimer topology is a viable strategy to "tune" signaling output. We utilized diabodies (DA) as surrogate ligands in a prototypical dimeric receptor-ligand system, the cytokine Erythropoietin (EPO) and its receptor (EpoR), to dimerize EpoR ectodomains in non-native architectures. Diabody-induced signaling amplitudes varied from full to minimal agonism, and structures of these DA/EpoR complexes differed in EpoR dimer orientation and proximity. Diabodies also elicited biased or differential activation of signaling pathways and gene expression profiles compared to EPO. Non-signaling diabodies inhibited proliferation of erythroid precursors from patients with a myeloproliferative neoplasm due to a constitutively active JAK2V617F mutation. Thus, intracellular oncogenic mutations causing ligand-independent receptor activation can be counteracted by extracellular ligands that re-orient receptors into inactive dimer topologies. This approach has broad applications for tuning signaling output for many dimeric receptor systems.
Asunto(s)
Receptores de Eritropoyetina/química , Receptores de Eritropoyetina/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Línea Celular , Cristalografía por Rayos X , Dimerización , Eritropoyetina/metabolismo , Humanos , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Ratones , Modelos Moleculares , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutación Puntual , Ingeniería de Proteínas , Receptores de Eritropoyetina/agonistas , Receptores de Eritropoyetina/antagonistas & inhibidores , Alineación de SecuenciaRESUMEN
Proteins, particularly viral proteins, can be multifunctional, but the mechanisms behind multifunctionality are not fully understood. Here, we illustrate through multiple crystal structures, biochemistry, and cellular microscopy that VP40 rearranges into different structures, each with a distinct function required for the ebolavirus life cycle. A butterfly-shaped VP40 dimer traffics to the cellular membrane. Once there, electrostatic interactions trigger rearrangement of the polypeptide into a linear hexamer. These hexamers construct a multilayered, filamentous matrix structure that is critical for budding and resembles tomograms of authentic virions. A third structure of VP40, formed by a different rearrangement, is not involved in virus assembly but instead uniquely binds RNA to regulate viral transcription inside infected cells. These results provide a functional model for ebolavirus matrix assembly and the other roles of VP40 in the virus life cycle and demonstrate how a single wild-type, unmodified polypeptide can assemble into different structures for different functions.
Asunto(s)
Ebolavirus/metabolismo , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/metabolismo , Cristalografía por Rayos X , Dimerización , Ebolavirus/química , Ebolavirus/clasificación , Ebolavirus/genética , Modelos Moleculares , Mutagénesis , Mutación Puntual , Proteínas de la Matriz Viral/genética , Ensamble de Virus , Liberación del VirusRESUMEN
RNA polymerase II (RNAPII) lies at the core of dynamic control of gene expression. Using 53 RNAPII point mutants, we generated a point mutant epistatic miniarray profile (pE-MAP) comprising â¼60,000 quantitative genetic interactions in Saccharomyces cerevisiae. This analysis enabled functional assignment of RNAPII subdomains and uncovered connections between individual regions and other protein complexes. Using splicing microarrays and mutants that alter elongation rates in vitro, we found an inverse relationship between RNAPII speed and in vivo splicing efficiency. Furthermore, the pE-MAP classified fast and slow mutants that favor upstream and downstream start site selection, respectively. The striking coordination of polymerization rate with transcription initiation and splicing suggests that transcription rate is tuned to regulate multiple gene expression steps. The pE-MAP approach provides a powerful strategy to understand other multifunctional machines at amino acid resolution.
Asunto(s)
Epistasis Genética , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Alelos , Estudio de Asociación del Genoma Completo , Mutación Puntual , ARN Polimerasa II/química , Empalme del ARN , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Sitio de Iniciación de la Transcripción , Transcripción Genética , TranscriptomaRESUMEN
The Polycomb repressive system is an essential chromatin-based regulator of gene expression. Despite being extensively studied, how the Polycomb system selects its target genes is poorly understood, and whether its histone-modifying activities are required for transcriptional repression remains controversial. Here, we directly test the requirement for PRC1 catalytic activity in Polycomb system function. To achieve this, we develop a conditional mutation system in embryonic stem cells that completely removes PRC1 catalytic activity. Using this system, we demonstrate that catalysis by PRC1 drives Polycomb chromatin domain formation and long-range chromatin interactions. Furthermore, we show that variant PRC1 complexes with DNA-binding activities occupy target sites independently of PRC1 catalytic activity, providing a putative mechanism for Polycomb target site selection. Finally, we discover that Polycomb-mediated gene repression requires PRC1 catalytic activity. Together these discoveries provide compelling evidence that PRC1 catalysis is central to Polycomb system function and gene regulation.
Asunto(s)
Regulación de la Expresión Génica , Complejo Represivo Polycomb 1/metabolismo , Animales , Biocatálisis , Línea Celular , Cromatina/metabolismo , Células Madre Embrionarias/enzimología , Células Madre Embrionarias/metabolismo , Células HEK293 , Histonas/metabolismo , Humanos , Masculino , Ratones , Mutación Puntual , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 2/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Cancer evolves dynamically as clonal expansions supersede one another driven by shifting selective pressures, mutational processes, and disrupted cancer genes. These processes mark the genome, such that a cancer's life history is encrypted in the somatic mutations present. We developed algorithms to decipher this narrative and applied them to 21 breast cancers. Mutational processes evolve across a cancer's lifespan, with many emerging late but contributing extensive genetic variation. Subclonal diversification is prominent, and most mutations are found in just a fraction of tumor cells. Every tumor has a dominant subclonal lineage, representing more than 50% of tumor cells. Minimal expansion of these subclones occurs until many hundreds to thousands of mutations have accumulated, implying the existence of long-lived, quiescent cell lineages capable of substantial proliferation upon acquisition of enabling genomic changes. Expansion of the dominant subclone to an appreciable mass may therefore represent the final rate-limiting step in a breast cancer's development, triggering diagnosis.
Asunto(s)
Neoplasias de la Mama/genética , Transformación Celular Neoplásica , Evolución Clonal , Mutación , Algoritmos , Aberraciones Cromosómicas , Femenino , Humanos , Mutación PuntualRESUMEN
We report the results of whole-genome and transcriptome sequencing of tumor and adjacent normal tissue samples from 17 patients with non-small cell lung carcinoma (NSCLC). We identified 3,726 point mutations and more than 90 indels in the coding sequence, with an average mutation frequency more than 10-fold higher in smokers than in never-smokers. Novel alterations in genes involved in chromatin modification and DNA repair pathways were identified, along with DACH1, CFTR, RELN, ABCB5, and HGF. Deep digital sequencing revealed diverse clonality patterns in both never-smokers and smokers. All validated EFGR and KRAS mutations were present in the founder clones, suggesting possible roles in cancer initiation. Analysis revealed 14 fusions, including ROS1 and ALK, as well as novel metabolic enzymes. Cell-cycle and JAK-STAT pathways are significantly altered in lung cancer, along with perturbations in 54 genes that are potentially targetable with currently available drugs.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Fumar/genética , Fumar/patología , Carcinoma de Pulmón de Células no Pequeñas/terapia , Aberraciones Cromosómicas , Femenino , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación INDEL , Neoplasias Pulmonares/terapia , Masculino , Terapia Molecular Dirigida , Mutación Puntual , Proteína ReelinaRESUMEN
A long-standing question in the study of long-term memory is how a memory trace persists for years when the proteins that initiated the process turn over and disappear within days. Previously, we postulated that self-sustaining amyloidogenic oligomers of cytoplasmic polyadenylation element-binding protein (CPEB) provide a mechanism for the maintenance of activity-dependent synaptic changes and, thus, the persistence of memory. Here, we found that the Drosophila CPEB Orb2 forms amyloid-like oligomers, and oligomers are enriched in the synaptic membrane fraction. Of the two protein isoforms of Orb2, the amyloid-like oligomer formation is dependent on the Orb2A form. A point mutation in the prion-like domain of Orb2A, which reduced amyloid-like oligomerization of Orb2, did not interfere with learning or memory persisting up to 24 hr. However the mutant flies failed to stabilize memory beyond 48 hr. These results support the idea that amyloid-like oligomers of neuronal CPEB are critical for the persistence of long-term memory.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Factores de Transcripción/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Secuencia de Aminoácidos , Amiloide/metabolismo , Animales , Aplysia/metabolismo , Encéfalo/metabolismo , Proteínas de Drosophila/química , Memoria , Datos de Secuencia Molecular , Neuronas/metabolismo , Mutación Puntual , Isoformas de Proteínas/metabolismo , Sinapsis/metabolismo , Factores de Transcripción/química , Factores de Escisión y Poliadenilación de ARNm/químicaRESUMEN
The mutation and overexpression of the epidermal growth factor receptor (EGFR) are associated with the development of a variety of cancers, making this prototypical dimerization-activated receptor tyrosine kinase a prominent target of cancer drugs. Using long-timescale molecular dynamics simulations, we find that the N lobe dimerization interface of the wild-type EGFR kinase domain is intrinsically disordered and that it becomes ordered only upon dimerization. Our simulations suggest, moreover, that some cancer-linked mutations distal to the dimerization interface, particularly the widespread L834R mutation (also referred to as L858R), facilitate EGFR dimerization by suppressing this local disorder. Corroborating these findings, our biophysical experiments and kinase enzymatic assays indicate that the L834R mutation causes abnormally high activity primarily by promoting EGFR dimerization rather than by allowing activation without dimerization. We also find that phosphorylation of EGFR kinase domain at Tyr845 may suppress the intrinsic disorder, suggesting a molecular mechanism for autonomous EGFR signaling.
Asunto(s)
Receptores ErbB/química , Receptores ErbB/genética , Neoplasias/metabolismo , Mutación Puntual , Transducción de Señal , Secuencia de Aminoácidos , Cristalografía por Rayos X , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Gefitinib , Humanos , Lapatinib , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Pliegue de Proteína , Inhibidores de Proteínas Quinasas/farmacología , Multimerización de Proteína , Estructura Terciaria de Proteína , Quinazolinas/farmacología , Alineación de SecuenciaRESUMEN
Molecular switch proteins whose cycling between states is controlled by opposing regulators1,2 are central to biological signal transduction. As switch proteins function within highly connected interaction networks3, the fundamental question arises of how functional specificity is achieved when different processes share common regulators. Here we show that functional specificity of the small GTPase switch protein Gsp1 in Saccharomyces cerevisiae (the homologue of the human protein RAN)4 is linked to differential sensitivity of biological processes to different kinetics of the Gsp1 (RAN) switch cycle. We make 55 targeted point mutations to individual protein interaction interfaces of Gsp1 (RAN) and show through quantitative genetic5 and physical interaction mapping that Gsp1 (RAN) interface perturbations have widespread cellular consequences. Contrary to expectation, the cellular effects of the interface mutations group by their biophysical effects on kinetic parameters of the GTPase switch cycle and not by the targeted interfaces. Instead, we show that interface mutations allosterically tune the GTPase cycle kinetics. These results suggest a model in which protein partner binding, or post-translational modifications at distal sites, could act as allosteric regulators of GTPase switching. Similar mechanisms may underlie regulation by other GTPases, and other biological switches. Furthermore, our integrative platform to determine the quantitative consequences of molecular perturbations may help to explain the effects of disease mutations that target central molecular switches.