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1.
Cell ; 187(10): 2446-2464.e22, 2024 May 09.
Artículo en Inglés | MEDLINE | ID: mdl-38582079

RESUMEN

Tauopathies are age-associated neurodegenerative diseases whose mechanistic underpinnings remain elusive, partially due to a lack of appropriate human models. Here, we engineered human induced pluripotent stem cell (hiPSC)-derived neuronal lines to express 4R Tau and 4R Tau carrying the P301S MAPT mutation when differentiated into neurons. 4R-P301S neurons display progressive Tau inclusions upon seeding with Tau fibrils and recapitulate features of tauopathy phenotypes including shared transcriptomic signatures, autophagic body accumulation, and reduced neuronal activity. A CRISPRi screen of genes associated with Tau pathobiology identified over 500 genetic modifiers of seeding-induced Tau propagation, including retromer VPS29 and genes in the UFMylation cascade. In progressive supranuclear palsy (PSP) and Alzheimer's Disease (AD) brains, the UFMylation cascade is altered in neurofibrillary-tangle-bearing neurons. Inhibiting the UFMylation cascade in vitro and in vivo suppressed seeding-induced Tau propagation. This model provides a robust platform to identify novel therapeutic strategies for 4R tauopathy.


Asunto(s)
Células Madre Pluripotentes Inducidas , Neuronas , Tauopatías , Proteínas tau , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas tau/metabolismo , Tauopatías/metabolismo , Tauopatías/patología , Neuronas/metabolismo , Neuronas/patología , Animales , Ratones , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/genética , Encéfalo/metabolismo , Encéfalo/patología , Parálisis Supranuclear Progresiva/metabolismo , Parálisis Supranuclear Progresiva/patología , Parálisis Supranuclear Progresiva/genética , Diferenciación Celular , Mutación , Autofagia
2.
Cell ; 186(2): 346-362.e17, 2023 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-36638793

RESUMEN

Ribosomes frequently stall during mRNA translation, resulting in the context-dependent activation of quality control pathways to maintain proteostasis. However, surveillance mechanisms that specifically respond to stalled ribosomes with an occluded A site have not been identified. We discovered that the elongation factor-1α (eEF1A) inhibitor, ternatin-4, triggers the ubiquitination and degradation of eEF1A on stalled ribosomes. Using a chemical genetic approach, we unveiled a signaling network comprising two E3 ligases, RNF14 and RNF25, which are required for eEF1A degradation. Quantitative proteomics revealed the RNF14 and RNF25-dependent ubiquitination of eEF1A and a discrete set of ribosomal proteins. The ribosome collision sensor GCN1 plays an essential role by engaging RNF14, which directly ubiquitinates eEF1A. The site-specific, RNF25-dependent ubiquitination of the ribosomal protein RPS27A/eS31 provides a second essential signaling input. Our findings illuminate a ubiquitin signaling network that monitors the ribosomal A site and promotes the degradation of stalled translation factors, including eEF1A and the termination factor eRF1.


Asunto(s)
Proteínas de Unión al ARN , Transactivadores , Proteínas Portadoras/metabolismo , Factores de Elongación de Péptidos/genética , Biosíntesis de Proteínas , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Humanos , Células HeLa , Células HEK293 , Proteínas de Unión al ARN/metabolismo , Transactivadores/metabolismo , Factor 1 de Elongación Peptídica/metabolismo
3.
Cell ; 184(9): 2503-2519.e17, 2021 04 29.
Artículo en Inglés | MEDLINE | ID: mdl-33838111

RESUMEN

A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff-a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.


Asunto(s)
Sistemas CRISPR-Cas , Reprogramación Celular , Epigénesis Genética , Epigenoma , Edición Génica , Células Madre Pluripotentes Inducidas/citología , Neuronas/citología , Diferenciación Celular , Islas de CpG , Metilación de ADN , Silenciador del Gen , Código de Histonas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/metabolismo , Procesamiento Proteico-Postraduccional
4.
Cell ; 174(4): 953-967.e22, 2018 08 09.
Artículo en Inglés | MEDLINE | ID: mdl-30033366

RESUMEN

Seminal yeast studies have established the value of comprehensively mapping genetic interactions (GIs) for inferring gene function. Efforts in human cells using focused gene sets underscore the utility of this approach, but the feasibility of generating large-scale, diverse human GI maps remains unresolved. We developed a CRISPR interference platform for large-scale quantitative mapping of human GIs. We systematically perturbed 222,784 gene pairs in two cancer cell lines. The resultant maps cluster functionally related genes, assigning function to poorly characterized genes, including TMEM261, a new electron transport chain component. Individual GIs pinpoint unexpected relationships between pathways, exemplified by a specific cholesterol biosynthesis intermediate whose accumulation induces deoxynucleotide depletion, causing replicative DNA damage and a synthetic-lethal interaction with the ATR/9-1-1 DNA repair pathway. Our map provides a broad resource, establishes GI maps as a high-resolution tool for dissecting gene function, and serves as a blueprint for mapping the genetic landscape of human cells.


Asunto(s)
Biomarcadores/metabolismo , Colesterol/metabolismo , Epistasis Genética , Redes Reguladoras de Genes , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células Jurkat , Células K562 , Mapeo de Interacción de Proteínas
5.
Cell ; 174(3): 505-520, 2018 07 26.
Artículo en Inglés | MEDLINE | ID: mdl-30053424

RESUMEN

Although gene discovery in neuropsychiatric disorders, including autism spectrum disorder, intellectual disability, epilepsy, schizophrenia, and Tourette disorder, has accelerated, resulting in a large number of molecular clues, it has proven difficult to generate specific hypotheses without the corresponding datasets at the protein complex and functional pathway level. Here, we describe one path forward-an initiative aimed at mapping the physical and genetic interaction networks of these conditions and then using these maps to connect the genomic data to neurobiology and, ultimately, the clinic. These efforts will include a team of geneticists, structural biologists, neurobiologists, systems biologists, and clinicians, leveraging a wide array of experimental approaches and creating a collaborative infrastructure necessary for long-term investigation. This initiative will ultimately intersect with parallel studies that focus on other diseases, as there is a significant overlap with genes implicated in cancer, infectious disease, and congenital heart defects.


Asunto(s)
Mapeo Cromosómico/métodos , Trastornos del Neurodesarrollo/genética , Biología de Sistemas/métodos , Redes Reguladoras de Genes/genética , Predisposición Genética a la Enfermedad/genética , Estudio de Asociación del Genoma Completo/métodos , Genómica/métodos , Humanos , Neurobiología/métodos , Neuropsiquiatría
6.
Mol Cell ; 82(3): 555-569.e7, 2022 02 03.
Artículo en Inglés | MEDLINE | ID: mdl-35063133

RESUMEN

In the eukaryotic cytosol, the Hsp70 and the Hsp90 chaperone machines work in tandem with the maturation of a diverse array of client proteins. The transfer of nonnative clients between these systems is essential to the chaperoning process, but how it is regulated is still not clear. We discovered that NudC is an essential transfer factor with an unprecedented mode of action: NudC interacts with Hsp40 in Hsp40-Hsp70-client complexes and displaces Hsp70. Then, the interaction of NudC with Hsp90 allows the direct transfer of Hsp40-bound clients to Hsp90 for further processing. Consistent with this mechanism, NudC increases client activation in vitro as well as in cells and is essential for cellular viability. Together, our results show the complexity of the cooperation between the major chaperone machineries in the eukaryotic cytosol.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas Nucleares/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular/genética , Supervivencia Celular , Células HEK293 , Proteínas del Choque Térmico HSP40/genética , Proteínas HSP90 de Choque Térmico/genética , Humanos , Células K562 , Cinética , Simulación del Acoplamiento Molecular , Proteínas Nucleares/genética , Unión Proteica , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo
7.
Nat Rev Neurosci ; 25(5): 351-371, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38575768

RESUMEN

The selective vulnerability of specific neuronal subtypes is a hallmark of neurodegenerative diseases. In this Review, I summarize our current understanding of the brain regions and cell types that are selectively vulnerable in different neurodegenerative diseases and describe the proposed underlying cell-autonomous and non-cell-autonomous mechanisms. I highlight how recent methodological innovations - including single-cell transcriptomics, CRISPR-based screens and human cell-based models of disease - are enabling new breakthroughs in our understanding of selective vulnerability. An understanding of the molecular mechanisms that determine selective vulnerability and resilience would shed light on the key processes that drive neurodegeneration and point to potential therapeutic strategies to protect vulnerable cell populations.


Asunto(s)
Enfermedades Neurodegenerativas , Humanos , Encéfalo/metabolismo , Encéfalo/patología , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Neuronas/metabolismo
8.
Cell ; 159(3): 647-61, 2014 Oct 23.
Artículo en Inglés | MEDLINE | ID: mdl-25307932

RESUMEN

While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%-99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a ∼1,000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways.


Asunto(s)
Sistemas CRISPR-Cas , Técnicas Genéticas , Transcripción Genética , Línea Celular , Toxina del Cólera/metabolismo , Toxina Diftérica/metabolismo , Genoma Humano , Humanos
9.
Cell ; 152(4): 909-22, 2013 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-23394947

RESUMEN

Genetic interaction (GI) maps, comprising pairwise measures of how strongly the function of one gene depends on the presence of a second, have enabled the systematic exploration of gene function in microorganisms. Here, we present a two-stage strategy to construct high-density GI maps in mammalian cells. First, we use ultracomplex pooled shRNA libraries (25 shRNAs/gene) to identify high-confidence hit genes for a given phenotype and effective shRNAs. We then construct double-shRNA libraries from these to systematically measure GIs between hits. A GI map focused on ricin susceptibility broadly recapitulates known pathways and provides many unexpected insights. These include a noncanonical role for COPI, a previously uncharacterized protein complex affecting toxin clearance, a specialized role for the ribosomal protein RPS25, and functionally distinct mammalian TRAPP complexes. The ability to rapidly generate mammalian GI maps provides a potentially transformative tool for defining gene function and designing combination therapies based on synergistic pairs.


Asunto(s)
Transporte Biológico , Epistasis Genética , Ricina/toxicidad , Atorvastatina , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Proteína Coat de Complejo I/metabolismo , Retículo Endoplásmico/metabolismo , Ácidos Heptanoicos/farmacología , Humanos , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Pirroles/farmacología , ARN Interferente Pequeño , Proteínas Ribosómicas/metabolismo , Proteínas de Transporte Vesicular/metabolismo
10.
Mol Cell ; 79(1): 191-198.e3, 2020 07 02.
Artículo en Inglés | MEDLINE | ID: mdl-32619469

RESUMEN

We recently used CRISPRi/a-based chemical-genetic screens and cell biological, biochemical, and structural assays to determine that rigosertib, an anti-cancer agent in phase III clinical trials, kills cancer cells by destabilizing microtubules. Reddy and co-workers (Baker et al., 2020, this issue of Molecular Cell) suggest that a contaminating degradation product in commercial formulations of rigosertib is responsible for the microtubule-destabilizing activity. Here, we demonstrate that cells treated with pharmaceutical-grade rigosertib (>99.9% purity) or commercially obtained rigosertib have qualitatively indistinguishable phenotypes across multiple assays. The two formulations have indistinguishable chemical-genetic interactions with genes that modulate microtubule stability, both destabilize microtubules in cells and in vitro, and expression of a rationally designed tubulin mutant with a mutation in the rigosertib binding site (L240F TUBB) allows cells to proliferate in the presence of either formulation. Importantly, the specificity of the L240F TUBB mutant for microtubule-destabilizing agents has been confirmed independently. Thus, rigosertib kills cancer cells by destabilizing microtubules, in agreement with our original findings.


Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular , Glicina/análogos & derivados , Microtúbulos/efectos de los fármacos , Neoplasias/patología , Preparaciones Farmacéuticas/metabolismo , Sulfonas/farmacología , Tubulina (Proteína)/metabolismo , Células Cultivadas , Cristalografía por Rayos X , Contaminación de Medicamentos , Glicina/farmacología , Humanos , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Preparaciones Farmacéuticas/química , Conformación Proteica , Tubulina (Proteína)/química , Tubulina (Proteína)/genética
11.
Mol Cell ; 74(1): 32-44.e8, 2019 04 04.
Artículo en Inglés | MEDLINE | ID: mdl-30846318

RESUMEN

Excessive levels of saturated fatty acids are toxic to cells, although the basis for this lipotoxicity remains incompletely understood. Here, we analyzed the transcriptome, lipidome, and genetic interactions of human leukemia cells exposed to palmitate. Palmitate treatment increased saturated glycerolipids, accompanied by a transcriptional stress response, including upregulation of the endoplasmic reticulum (ER) stress response. A comprehensive genome-wide short hairpin RNA (shRNA) screen identified >350 genes modulating lipotoxicity. Among previously unknown genetic modifiers of lipotoxicity, depletion of RNF213, a putative ubiquitin ligase mutated in Moyamoya vascular disease, protected cells from lipotoxicity. On a broader level, integration of our comprehensive datasets revealed that changes in di-saturated glycerolipids, but not other lipid classes, are central to lipotoxicity in this model. Consistent with this, inhibition of ER-localized glycerol-3-phosphate acyltransferase activity protected from all aspects of lipotoxicity. Identification of genes modulating the response to saturated fatty acids may reveal novel therapeutic strategies for treating metabolic diseases linked to lipotoxicity.


Asunto(s)
Estrés del Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Glicéridos/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Ácido Palmítico/toxicidad , Aciltransferasas/genética , Aciltransferasas/metabolismo , Adenosina Trifosfatasas/metabolismo , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Estrés del Retículo Endoplásmico/genética , Regulación Enzimológica de la Expresión Génica , Células HeLa , Células Hep G2 , Humanos , Células K562 , Metabolismo de los Lípidos/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Transcriptoma , Ubiquitina-Proteína Ligasas/metabolismo
12.
Proc Natl Acad Sci U S A ; 121(22): e2315690121, 2024 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-38781206

RESUMEN

The prion-like spread of protein aggregates is a leading hypothesis for the propagation of neurofibrillary lesions in the brain, including the spread of tau inclusions associated with Alzheimer's disease. The mechanisms of cellular uptake of tau seeds and subsequent nucleated polymerization of cytosolic tau are major questions in the field, and the potential for coupling between the entry and nucleation mechanisms has been little explored. We found that in primary astrocytes and neurons, endocytosis of tau seeds leads to their accumulation in lysosomes. This in turn leads to lysosomal swelling, deacidification, and recruitment of ESCRT proteins, but not Galectin-3, to the lysosomal membrane. These observations are consistent with nanoscale damage of the lysosomal membrane. Live cell imaging and STORM superresolution microscopy further show that the nucleation of cytosolic tau occurs primarily at the lysosome membrane under these conditions. These data suggest that tau seeds escape from lysosomes via nanoscale damage rather than wholesale rupture and that nucleation of cytosolic tau commences as soon as tau fibril ends emerge from the lysosomal membrane.


Asunto(s)
Citosol , Lisosomas , Proteínas tau , Proteínas tau/metabolismo , Lisosomas/metabolismo , Citosol/metabolismo , Animales , Astrocitos/metabolismo , Astrocitos/patología , Neuronas/metabolismo , Neuronas/patología , Humanos , Membranas Intracelulares/metabolismo , Endocitosis , Ratones , Células Cultivadas
13.
Nature ; 579(7799): 427-432, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32132707

RESUMEN

In mammalian cells, mitochondrial dysfunction triggers the integrated stress response, in which the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) results in the induction of the transcription factor ATF41-3. However, how mitochondrial stress is relayed to ATF4 is unknown. Here we show that HRI is the eIF2α kinase that is necessary and sufficient for this relay. In a genome-wide CRISPR interference screen, we identified factors upstream of HRI: OMA1, a mitochondrial stress-activated protease; and DELE1, a little-characterized protein that we found was associated with the inner mitochondrial membrane. Mitochondrial stress stimulates OMA1-dependent cleavage of DELE1 and leads to the accumulation of DELE1 in the cytosol, where it interacts with HRI and activates the eIF2α kinase activity of HRI. In addition, DELE1 is required for ATF4 translation downstream of eIF2α phosphorylation. Blockade of the OMA1-DELE1-HRI pathway triggers an alternative response in which specific molecular chaperones are induced. The OMA1-DELE1-HRI pathway therefore represents a potential therapeutic target that could enable fine-tuning of the integrated stress response for beneficial outcomes in diseases that involve mitochondrial dysfunction.


Asunto(s)
Citosol/metabolismo , Metaloendopeptidasas/metabolismo , Mitocondrias/metabolismo , Mitocondrias/patología , Proteínas Mitocondriales/metabolismo , Estrés Fisiológico , eIF-2 Quinasa/metabolismo , Factor de Transcripción Activador 4/biosíntesis , Factor de Transcripción Activador 4/metabolismo , Sistemas CRISPR-Cas , Línea Celular , Citosol/enzimología , Activación Enzimática , Factor 2 Eucariótico de Iniciación/metabolismo , Humanos , Masculino , Proteínas Mitocondriales/química , Chaperonas Moleculares/metabolismo , Fosforilación , Unión Proteica
14.
Nature ; 579(7797): 136-140, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32076268

RESUMEN

Metazoan development requires the robust proliferation of progenitor cells, the identities of which are established by tightly controlled transcriptional networks1. As gene expression is globally inhibited during mitosis, the transcriptional programs that define cell identity must be restarted in each cell cycle2-5 but how this is accomplished is poorly understood. Here we identify a ubiquitin-dependent mechanism that integrates gene expression with cell division to preserve cell identity. We found that WDR5 and TBP, which bind active interphase promoters6,7, recruit the anaphase-promoting complex (APC/C) to specific transcription start sites during mitosis. This allows APC/C to decorate histones with ubiquitin chains branched at Lys11 and Lys48 (K11/K48-branched ubiquitin chains) that recruit p97 (also known as VCP) and the proteasome, which ensures the rapid expression of pluripotency genes in the next cell cycle. Mitotic exit and the re-initiation of transcription are thus controlled by a single regulator (APC/C), which provides a robust mechanism for maintaining cell identity throughout cell division.


Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Diferenciación Celular/genética , Regulación de la Expresión Génica , Complejos Multiproteicos/metabolismo , Anafase , División Celular , Células HEK293 , Células HeLa , Histonas/química , Histonas/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Interfase , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mitosis , Organofosfatos/metabolismo , Poliubiquitina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Sitio de Iniciación de la Transcripción , Ubiquitina/metabolismo , Ubiquitinación
15.
Nature ; 580(7803): 381-385, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32296178

RESUMEN

The spread of protein aggregates during disease progression is a common theme underlying many neurodegenerative diseases. The microtubule-associated protein tau has a central role in the pathogenesis of several forms of dementia known as tauopathies-including Alzheimer's disease, frontotemporal dementia and chronic traumatic encephalopathy1. Progression of these diseases is characterized by the sequential spread and deposition of protein aggregates in a predictable pattern that correlates with clinical severity2. This observation and complementary experimental studies3,4 have suggested that tau can spread in a prion-like manner, by passing to naive cells in which it templates misfolding and aggregation. However, although the propagation of tau has been extensively studied, the underlying cellular mechanisms remain poorly understood. Here we show that the low-density lipoprotein receptor-related protein 1 (LRP1) controls the endocytosis of tau and its subsequent spread. Knockdown of LRP1 significantly reduced tau uptake in H4 neuroglioma cells and in induced pluripotent stem cell-derived neurons. The interaction between tau and LRP1 is mediated by lysine residues in the microtubule-binding repeat region of tau. Furthermore, downregulation of LRP1 in an in vivo mouse model of tau spread was found to effectively reduce the propagation of tau between neurons. Our results identify LRP1 as a key regulator of tau spread in the brain, and therefore a potential target for the treatment of diseases that involve tau spread and aggregation.


Asunto(s)
Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteínas tau/metabolismo , Animales , Línea Celular , Endocitosis , Femenino , Humanos , Ligandos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Masculino , Ratones , Neuronas/metabolismo
16.
Mol Cell ; 68(1): 210-223.e6, 2017 Oct 05.
Artículo en Inglés | MEDLINE | ID: mdl-28985505

RESUMEN

Chemical libraries paired with phenotypic screens can now readily identify compounds with therapeutic potential. A central limitation to exploiting these compounds, however, has been in identifying their relevant cellular targets. Here, we present a two-tiered CRISPR-mediated chemical-genetic strategy for target identification: combined genome-wide knockdown and overexpression screening as well as focused, comparative chemical-genetic profiling. Application of these strategies to rigosertib, a drug in phase 3 clinical trials for high-risk myelodysplastic syndrome whose molecular target had remained controversial, pointed singularly to microtubules as rigosertib's target. We showed that rigosertib indeed directly binds to and destabilizes microtubules using cell biological, in vitro, and structural approaches. Finally, expression of tubulin with a structure-guided mutation in the rigosertib-binding pocket conferred resistance to rigosertib, establishing that rigosertib kills cancer cells by destabilizing microtubules. These results demonstrate the power of our chemical-genetic screening strategies for pinpointing the physiologically relevant targets of chemical agents.


Asunto(s)
Antineoplásicos/farmacología , Regulación Neoplásica de la Expresión Génica , Pruebas Genéticas/métodos , Glicina/análogos & derivados , Microtúbulos/efectos de los fármacos , Sulfonas/farmacología , Moduladores de Tubulina/farmacología , Tubulina (Proteína)/genética , Antineoplásicos/química , Sistemas CRISPR-Cas , Colchicina/farmacología , Resistencia a Antineoplásicos , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glicina/química , Glicina/farmacología , Células HeLa , Humanos , Células K562 , Cinesinas/genética , Cinesinas/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Mutación , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/metabolismo , Síndromes Mielodisplásicos/patología , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Sulfonas/química , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Vinblastina/farmacología
17.
J Cell Sci ; 135(4)2022 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-35188214

RESUMEN

February is LGBT+ history month, and to celebrate, Journal of Cell Science Editorial Advisory Board member David Bryant organised a conversation with a selection of scientists to explore their experiences of being LGBT+ in academia.


Asunto(s)
Liderazgo , Minorías Sexuales y de Género , Movilidad Laboral , Comunicación , Humanos
18.
J Neuroinflammation ; 21(1): 198, 2024 Aug 08.
Artículo en Inglés | MEDLINE | ID: mdl-39118084

RESUMEN

Astrocytes respond and contribute to neuroinflammation by adopting inflammatory reactive states. Although recent efforts have characterized the gene expression signatures associated with these reactive states, the cell biology underlying inflammatory reactive astrocyte phenotypes remains under-explored. Here, we used CRISPR-based screening in human iPSC-derived astrocytes to identify mTOR activation a driver of cytokine-induced endolysosomal system remodeling, manifesting as alkalinization of endolysosomal compartments, decreased autophagic flux, and increased exocytosis of certain endolysosomal cargos. Through endolysosomal proteomics, we identified and focused on one such cargo-IL-32, a disease-associated pro-inflammatory cytokine not present in rodents, whose secretion mechanism is not well understood. We found that IL-32 was partially secreted in extracellular vesicles likely to be exosomes. Furthermore, we found that IL-32 was involved in the polarization of inflammatory reactive astrocyte states and was upregulated in astrocytes in multiple sclerosis lesions. We believe that our results advance our understanding of cell biological pathways underlying inflammatory reactive astrocyte phenotypes and identify potential therapeutic targets.


Asunto(s)
Astrocitos , Exosomas , Interleucinas , Lisosomas , Serina-Treonina Quinasas TOR , Astrocitos/metabolismo , Humanos , Exosomas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Lisosomas/metabolismo , Interleucinas/metabolismo , Endosomas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Células Cultivadas , Enfermedades Neuroinflamatorias/metabolismo , Enfermedades Neuroinflamatorias/patología , Inflamación/metabolismo , Inflamación/patología
19.
PLoS Genet ; 16(10): e1009103, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-33052901

RESUMEN

G protein-coupled receptors (GPCRs) allow cells to respond to chemical and sensory stimuli through generation of second messengers, such as cyclic AMP (cAMP), which in turn mediate a myriad of processes, including cell survival, proliferation, and differentiation. In order to gain deeper insights into the complex biology and physiology of these key cellular pathways, it is critical to be able to globally map the molecular factors that shape cascade function. Yet, to this date, efforts to systematically identify regulators of GPCR/cAMP signaling have been lacking. Here, we combined genome-wide screening based on CRISPR interference with a novel sortable transcriptional reporter that provides robust readout for cAMP signaling, and carried out a functional screen for regulators of the pathway. Due to the sortable nature of the platform, we were able to assay regulators with strong and moderate phenotypes by analyzing sgRNA distribution among three fractions with distinct reporter expression. We identified 45 regulators with strong and 50 regulators with moderate phenotypes not previously known to be involved in cAMP signaling. In follow-up experiments, we validated the functional effects of seven newly discovered mediators (NUP93, PRIM1, RUVBL1, PKMYT1, TP53, SF3A2, and HRAS), and showed that they control distinct steps of the pathway. Thus, our study provides proof of principle that the screening platform can be applied successfully to identify bona fide regulators of GPCR/second messenger cascades in an unbiased and high-throughput manner, and illuminates the remarkable functional diversity among GPCR regulators.


Asunto(s)
Sistemas CRISPR-Cas/genética , Proliferación Celular/genética , AMP Cíclico/genética , Receptores Acoplados a Proteínas G/genética , ATPasas Asociadas con Actividades Celulares Diversas/genética , Proteínas Portadoras/genética , Diferenciación Celular/genética , Células Cultivadas , ADN Helicasas/genética , ADN Primasa/genética , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Proteínas de Complejo Poro Nuclear/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Factores de Empalme de ARN/genética , Transducción de Señal/genética , Proteína p53 Supresora de Tumor/genética
20.
Nat Chem Biol ; 16(6): 653-659, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32152544

RESUMEN

Defining the biologically active structures of proteins in their cellular environments remains challenging for proteins with multiple conformations and functions, where only a minor conformer might be associated with a given function. Here, we use deep mutational scanning to probe the structure and dynamics of α-synuclein, a protein known to adopt disordered, helical and amyloid conformations. We examined the effects of 2,600 single-residue substitutions on the ability of intracellularly expressed α-synuclein to slow the growth of yeast. Computational analysis of the data showed that the conformation responsible for this phenotype is a long, uninterrupted, amphiphilic helix with increasing dynamics toward the C terminus. Deep mutational scanning can therefore determine biologically active conformations in cellular environments, even for a highly dynamic multi-conformational protein.


Asunto(s)
Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutación , alfa-Sinucleína/química , alfa-Sinucleína/genética , Secuencia de Aminoácidos , Amiloide/química , Biblioteca Genómica , Modelos Moleculares , Fenotipo , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Levaduras/metabolismo
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