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1.
Cell ; 184(25): 6037-6051.e14, 2021 12 09.
Artículo en Inglés | MEDLINE | ID: mdl-34852237

RESUMEN

RNA viruses generate defective viral genomes (DVGs) that can interfere with replication of the parental wild-type virus. To examine their therapeutic potential, we created a DVG by deleting the capsid-coding region of poliovirus. Strikingly, intraperitoneal or intranasal administration of this genome, which we termed eTIP1, elicits an antiviral response, inhibits replication, and protects mice from several RNA viruses, including enteroviruses, influenza, and SARS-CoV-2. While eTIP1 replication following intranasal administration is limited to the nasal cavity, its antiviral action extends non-cell-autonomously to the lungs. eTIP1 broad-spectrum antiviral effects are mediated by both local and distal type I interferon responses. Importantly, while a single eTIP1 dose protects animals from SARS-CoV-2 infection, it also stimulates production of SARS-CoV-2 neutralizing antibodies that afford long-lasting protection from SARS-CoV-2 reinfection. Thus, eTIP1 is a safe and effective broad-spectrum antiviral generating short- and long-term protection against SARS-CoV-2 and other respiratory infections in animal models.


Asunto(s)
Proteínas de la Cápside/genética , Virus Interferentes Defectuosos/metabolismo , Replicación Viral/efectos de los fármacos , Administración Intranasal , Animales , Antivirales/farmacología , Anticuerpos ampliamente neutralizantes/inmunología , Anticuerpos ampliamente neutralizantes/farmacología , COVID-19 , Proteínas de la Cápside/metabolismo , Línea Celular , Virus Interferentes Defectuosos/patogenicidad , Modelos Animales de Enfermedad , Genoma Viral/genética , Humanos , Gripe Humana , Interferones/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Poliovirus/genética , Poliovirus/metabolismo , Infecciones del Sistema Respiratorio/virología , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/patogenicidad
2.
Nature ; 596(7873): 558-564, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34408324

RESUMEN

Viral pathogens are an ongoing threat to public health worldwide. Analysing their dependence on host biosynthetic pathways could lead to effective antiviral therapies1. Here we integrate proteomic analyses of polysomes with functional genomics and pharmacological interventions to define how enteroviruses and flaviviruses remodel host polysomes to synthesize viral proteins and disable host protein production. We find that infection with polio, dengue or Zika virus markedly modifies polysome composition, without major changes to core ribosome stoichiometry. These viruses use different strategies to evict a common set of translation initiation and RNA surveillance factors from polysomes while recruiting host machineries that are specifically required for viral biogenesis. Targeting these specialized viral polysomes could provide a new approach for antiviral interventions. For example, we find that both Zika and dengue use the collagen proline hydroxylation machinery to mediate cotranslational modification of conserved proline residues in the viral polyprotein. Genetic or pharmacological inhibition of proline hydroxylation impairs nascent viral polyprotein folding and induces its aggregation and degradation. Notably, such interventions prevent viral polysome remodelling and lower virus production. Our findings delineate the modular nature of polysome specialization at the virus-host interface and establish a powerful strategy to identify targets for selective antiviral interventions.


Asunto(s)
Flavivirus/crecimiento & desarrollo , Flavivirus/metabolismo , Interacciones Huésped-Patógeno , Hidroxilación , Procolágeno-Prolina Dioxigenasa/metabolismo , Prolina/metabolismo , Biosíntesis de Proteínas , Línea Celular , Colágeno/química , Colágeno/metabolismo , Virus del Dengue/genética , Virus del Dengue/crecimiento & desarrollo , Flavivirus/química , Regulación Viral de la Expresión Génica , Genómica , Factores Celulares Derivados del Huésped/antagonistas & inhibidores , Factores Celulares Derivados del Huésped/metabolismo , Interacciones Huésped-Patógeno/genética , Humanos , Sitios Internos de Entrada al Ribosoma , Chaperonas Moleculares/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Poliovirus/genética , Poliovirus/crecimiento & desarrollo , Polirribosomas/química , Polirribosomas/metabolismo , Agregado de Proteínas , Pliegue de Proteína , Mapas de Interacción de Proteínas , Proteolisis , Proteómica , Virus Zika/genética , Virus Zika/crecimiento & desarrollo
3.
PLoS Pathog ; 20(2): e1011535, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38335237

RESUMEN

A better mechanistic understanding of virus-host dependencies can help reveal vulnerabilities and identify opportunities for therapeutic intervention. Of particular interest are essential interactions that enable production of viral proteins, as those could target an early step in the virus lifecycle. Here, we use subcellular proteomics, ribosome profiling analyses and reporter assays to detect changes in protein synthesis dynamics during SARS-CoV-2 (CoV2) infection. We identify specific translation factors and molecular chaperones that are used by CoV2 to promote the synthesis and maturation of its own proteins. These can be targeted to inhibit infection, without major toxicity to the host. We also find that CoV2 non-structural protein 1 (Nsp1) cooperates with initiation factors EIF1 and 1A to selectively enhance translation of viral RNA. When EIF1/1A are depleted, more ribosomes initiate translation from a conserved upstream CUG start codon found in all genomic and subgenomic viral RNAs. This results in higher translation of an upstream open reading frame (uORF1) and lower translation of the main ORF, altering the stoichiometry of viral proteins and attenuating infection. Replacing the upstream CUG with AUG strongly inhibits translation of the main ORF independently of Nsp1, EIF1, or EIF1A. Taken together, our work describes multiple dependencies of CoV2 on host biosynthetic networks and proposes a model for dosage control of viral proteins through Nsp1-mediated control of translation start site selection.


Asunto(s)
COVID-19 , ARN Viral , Humanos , ARN Viral/genética , SARS-CoV-2/genética , COVID-19/genética , Factores de Iniciación de Péptidos , Proteínas Virales
5.
Genes Dev ; 27(16): 1834-44, 2013 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-23934657

RESUMEN

Monitoring protein synthesis is essential to our understanding of gene expression regulation, as protein abundance is thought to be predominantly controlled at the level of translation. Mass-spectrometric and RNA sequencing methods have been recently developed for investigating mRNA translation at a global level, but these still involve technical limitations and are not widely applicable. In this study, we describe a novel system-wide proteomic approach for direct monitoring of translation, termed puromycin-associated nascent chain proteomics (PUNCH-P), which is based on incorporation of biotinylated puromycin into newly synthesized proteins under cell-free conditions followed by streptavidin affinity purification and liquid chromatography-tandem mass spectrometry analysis. Using PUNCH-P, we measured cell cycle-specific fluctuations in synthesis for >5000 proteins in mammalian cells, identified proteins not previously implicated in cell cycle processes, and generated the first translational profile of a whole mouse brain. This simple and economical technique is broadly applicable to any cell type and tissue, enabling the identification and quantification of rapid proteome responses under various biological conditions.


Asunto(s)
Ciclo Celular/genética , Regulación del Desarrollo de la Expresión Génica , Proteómica/métodos , ARN Mensajero/metabolismo , Animales , Encéfalo/metabolismo , Células HeLa , Humanos , Espectrometría de Masas , Ratones , Proteínas/química , Proteómica/normas , Reproducibilidad de los Resultados , Ribosomas/metabolismo
6.
Nucleic Acids Res ; 45(10): 5945-5957, 2017 Jun 02.
Artículo en Inglés | MEDLINE | ID: mdl-28460002

RESUMEN

Precise regulation of mRNA translation is critical for proper cell division, but little is known about the factors that mediate it. To identify mRNA-binding proteins that regulate translation during mitosis, we analyzed the composition of polysomes from interphase and mitotic cells using unbiased quantitative mass-spectrometry (LC-MS/MS). We found that mitotic polysomes are enriched with a subset of proteins involved in RNA processing, including alternative splicing and RNA export. To demonstrate that these may indeed be regulators of translation, we focused on heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a test case and confirmed that it is recruited to elongating ribosomes during mitosis. Then, using a combination of pulsed SILAC, metabolic labeling and ribosome profiling, we showed that knockdown of hnRNP C affects both global and transcript-specific translation rates and found that hnRNP C is specifically important for translation of mRNAs that encode ribosomal proteins and translation factors. Taken together, our results demonstrate how proteomic analysis of polysomes can provide insight into translation regulation under various cellular conditions of interest and suggest that hnRNP C facilitates production of translation machinery components during mitosis to provide daughter cells with the ability to efficiently synthesize proteins as they enter G1 phase.


Asunto(s)
Mitosis/genética , Polirribosomas/fisiología , Biosíntesis de Proteínas/fisiología , Proteómica/métodos , Factores de Empalme de ARN/fisiología , Cromatografía Liquida , Fase G1 , Técnicas de Silenciamiento del Gen , Ontología de Genes , Células HeLa , Ribonucleoproteína Heterogénea-Nuclear Grupo C/antagonistas & inhibidores , Ribonucleoproteína Heterogénea-Nuclear Grupo C/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo C/fisiología , Humanos , Interfase , Interferencia de ARN , Factores de Empalme de ARN/aislamiento & purificación , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/aislamiento & purificación , Proteínas de Unión al ARN/fisiología , Proteínas Ribosómicas/genética , Espectrometría de Masas en Tándem
7.
PLoS Genet ; 11(10): e1005554, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26439921

RESUMEN

Studying the complex relationship between transcription, translation and protein degradation is essential to our understanding of biological processes in health and disease. The limited correlations observed between mRNA and protein abundance suggest pervasive regulation of post-transcriptional steps and support the importance of profiling mRNA levels in parallel to protein synthesis and degradation rates. In this work, we applied an integrative multi-omic approach to study gene expression along the mammalian cell cycle through side-by-side analysis of mRNA, translation and protein levels. Our analysis sheds new light on the significant contribution of both protein synthesis and degradation to the variance in protein expression. Furthermore, we find that translation regulation plays an important role at S-phase, while progression through mitosis is predominantly controlled by changes in either mRNA levels or protein stability. Specific molecular functions are found to be co-regulated and share similar patterns of mRNA, translation and protein expression along the cell cycle. Notably, these include genes and entire pathways not previously implicated in cell cycle progression, demonstrating the potential of this approach to identify novel regulatory mechanisms beyond those revealed by traditional expression profiling. Through this three-level analysis, we characterize different mechanisms of gene expression, discover new cycling gene products and highlight the importance and utility of combining datasets generated using different techniques that monitor distinct steps of gene expression.


Asunto(s)
Biosíntesis de Proteínas , Proteolisis , ARN Mensajero/biosíntesis , Transcripción Genética , División Celular/genética , Regulación de la Expresión Génica , Células HeLa , Humanos , Biosíntesis de Proteínas/genética , Proteoma/genética , ARN Mensajero/genética , Transcriptoma/genética
8.
Nat Cell Biol ; 26(6): 892-902, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38741019

RESUMEN

Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of a CAG trinucleotide repeat in the Huntingtin (HTT) gene, encoding a homopolymeric polyglutamine (polyQ) tract. Although mutant HTT (mHTT) protein is known to aggregate, the links between aggregation and neurotoxicity remain unclear. Here we show that both translation and aggregation of wild-type HTT and mHTT are regulated by a stress-responsive upstream open reading frame and that polyQ expansions cause abortive translation termination and release of truncated, aggregation-prone mHTT fragments. Notably, we find that mHTT depletes translation elongation factor eIF5A in brains of symptomatic HD mice and cultured HD cells, leading to pervasive ribosome pausing and collisions. Loss of eIF5A disrupts homeostatic controls and impairs recovery from acute stress. Importantly, drugs that inhibit translation initiation reduce premature termination and mitigate this escalating cascade of ribotoxic stress and dysfunction in HD.


Asunto(s)
Factor 5A Eucariótico de Iniciación de Traducción , Proteína Huntingtina , Enfermedad de Huntington , Factores de Iniciación de Péptidos , Péptidos , Proteostasis , Proteínas de Unión al ARN , Ribosomas , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Animales , Péptidos/metabolismo , Péptidos/genética , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Humanos , Ribosomas/metabolismo , Ribosomas/genética , Factores de Iniciación de Péptidos/metabolismo , Factores de Iniciación de Péptidos/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Ratones , Ratones Transgénicos , Modelos Animales de Enfermedad , Estrés Fisiológico , Encéfalo/metabolismo , Encéfalo/patología , Expansión de Repetición de Trinucleótido/genética
9.
bioRxiv ; 2023 Jul 06.
Artículo en Inglés | MEDLINE | ID: mdl-37461541

RESUMEN

A better mechanistic understanding of virus-host interactions can help reveal vulnerabilities and identify opportunities for therapeutic interventions. Of particular interest are essential interactions that enable production of viral proteins, as those could target an early step in the virus lifecycle. Here, we use subcellular proteomics, ribosome profiling analyses and reporter assays to detect changes in polysome composition and protein synthesis during SARS-CoV-2 (CoV2) infection. We identify specific translation factors and molecular chaperones whose inhibition impairs infectious particle production without major toxicity to the host. We find that CoV2 non-structural protein Nsp1 selectively enhances virus translation through functional interactions with initiation factor EIF1A. When EIF1A is depleted, more ribosomes initiate translation from an upstream CUG start codon, inhibiting translation of non-structural genes and reducing viral titers. Together, our work describes multiple dependencies of CoV2 on host biosynthetic networks and identifies druggable targets for potential antiviral development.

10.
Nat Commun ; 14(1): 692, 2023 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-36754966

RESUMEN

Huntington's disease (HD) is caused by an expanded CAG repeat in the huntingtin gene, yielding a Huntingtin protein with an expanded polyglutamine tract. While experiments with patient-derived induced pluripotent stem cells (iPSCs) can help understand disease, defining pathological biomarkers remains challenging. Here, we used cryogenic electron tomography to visualize neurites in HD patient iPSC-derived neurons with varying CAG repeats, and primary cortical neurons from BACHD, deltaN17-BACHD, and wild-type mice. In HD models, we discovered sheet aggregates in double membrane-bound organelles, and mitochondria with distorted cristae and enlarged granules, likely mitochondrial RNA granules. We used artificial intelligence to quantify mitochondrial granules, and proteomics experiments reveal differential protein content in isolated HD mitochondria. Knockdown of Protein Inhibitor of Activated STAT1 ameliorated aberrant phenotypes in iPSC- and BACHD neurons. We show that integrated ultrastructural and proteomic approaches may uncover early HD phenotypes to accelerate diagnostics and the development of targeted therapeutics for HD.


Asunto(s)
Enfermedad de Huntington , Células Madre Pluripotentes Inducidas , Animales , Ratones , Inteligencia Artificial , Modelos Animales de Enfermedad , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Mitocondrias/metabolismo , Neuronas/metabolismo , Fenotipo , Proteómica , Humanos
11.
J Biol Chem ; 286(32): 27927-35, 2011 Aug 12.
Artículo en Inglés | MEDLINE | ID: mdl-21665947

RESUMEN

Translation elongation in eukaryotes is mediated by the concerted actions of elongation factor 1A (eEF1A), which delivers aminoacylated tRNA to the ribosome; elongation factor 1B (eEF1B) complex, which catalyzes the exchange of GDP to GTP on eEF1A; and eEF2, which facilitates ribosomal translocation. Here we present evidence in support of a novel mode of translation regulation by hindered tRNA delivery during mitosis. A conserved consensus phosphorylation site for the mitotic cyclin-dependent kinase 1 on the catalytic delta subunit of eEF1B (termed eEF1D) is required for its posttranslational modification during mitosis, resulting in lower affinity to its substrate eEF1A. This modification is correlated with reduced availability of eEF1A·tRNA complexes, as well as reduced delivery of tRNA to and association of eEF1A with elongating ribosomes. This mode of regulation by hindered tRNA delivery, although first discovered in mitosis, may represent a more globally applicable mechanism employed under other physiological conditions that involve down-regulation of protein synthesis at the elongation level.


Asunto(s)
Proteína Quinasa CDC2/metabolismo , Mitosis/fisiología , Extensión de la Cadena Peptídica de Translación/fisiología , Factor 1 de Elongación Peptídica/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Proteína Quinasa CDC2/genética , Células HeLa , Humanos , Factor 1 de Elongación Peptídica/genética , Factor 2 de Elongación Peptídica/genética , Factor 2 de Elongación Peptídica/metabolismo , Fosforilación/fisiología , ARN de Transferencia/genética , Ribosomas/genética
12.
Comput Struct Biotechnol J ; 18: 1074-1083, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32435426

RESUMEN

Puromycin is a naturally occurring aminonucleoside antibiotic that inhibits protein synthesis by ribosome-catalyzed incorporation into the C-terminus of elongating nascent chains, blocking further extension and resulting in premature termination of translation. It is most commonly known as a selection marker for cell lines genetically engineered to express a resistance transgene, but its additional uses as a probe for protein synthesis have proven invaluable across a wide variety of model systems, ranging from purified ribosomes and cell-free translation to intact cultured cells and whole animals. Puromycin is comprised of a nucleoside covalently bound to an amino acid, mimicking the 3' end of aminoacylated tRNAs that participate in delivery of amino acids to elongating ribosomes. Both moieties can tolerate some chemical substitutions and modifications without significant loss of activity, generating a diverse toolbox of puromycin-based reagents with added functionality, such as biotin for affinity purification or fluorophores for fluorescent microscopy detection. These reagents, as well as anti-puromycin antibodies, have played a pivotal role in advancing our understanding of the regulation and dysregulation of protein synthesis in normal and pathological processes, including immune response and neurological function. This manuscript reviews the current state of puromycin-based research, including structure and mechanism of action, relevant derivatives, use in advanced methodologies and some of the major insights generated using such techniques both in the lab and the clinic.

13.
Artículo en Inglés | MEDLINE | ID: mdl-30858229

RESUMEN

Viruses are obligate intracellular parasites that rely on their hosts for protein synthesis, genome replication, and viral particle production. As such, they have evolved mechanisms to divert host resources, including molecular chaperones, facilitate folding and assembly of viral proteins, stabilize complex structures under constant mutational pressure, and modulate signaling pathways to dampen antiviral responses and prevent premature host death. Biogenesis of viral proteins often presents unique challenges to the proteostasis network, as it requires the rapid and orchestrated production of high levels of a limited number of multifunctional, multidomain, and aggregation-prone proteins. To overcome such challenges, viruses interact with the folding machinery not only as clients but also as regulators of chaperone expression, function, and subcellular localization. In this review, we summarize the main types of interactions between viral proteins and chaperones during infection, examine evolutionary aspects of this relationship, and discuss the potential of using chaperone inhibitors as broad-spectrum antivirals.


Asunto(s)
Chaperonas Moleculares/metabolismo , Replicación Viral/fisiología , Virus/metabolismo , Transporte Activo de Núcleo Celular , Animales , Antivirales/farmacología , Apoptosis , Cápside , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Ratones , Oxígeno/química , Pliegue de Proteína , Proteostasis , Transducción de Señal , Virosis
14.
Sci Rep ; 6: 21635, 2016 Feb 22.
Artículo en Inglés | MEDLINE | ID: mdl-26898226

RESUMEN

Two novel approaches were recently suggested for genome-wide identification of protein aspects synthesized at a given time. Ribo-Seq is based on sequencing all the ribosome protected mRNA fragments in a cell, while PUNCH-P is based on mass-spectrometric analysis of only newly synthesized proteins. Here we describe the first Ribo-Seq/PUNCH-P comparison via the analysis of mammalian cells during the cell-cycle for detecting relevant differentially expressed genes between G1 and M phase. Our analyses suggest that the two approaches significantly overlap with each other. However, we demonstrate that there are biologically meaningful proteins/genes that can be detected to be post-transcriptionally regulated during the mammalian cell cycle only by each of the approaches, or their consolidation. Such gene sets are enriched with proteins known to be related to intra-cellular signalling pathways such as central cell cycle processes, central gene expression regulation processes, processes related to chromosome segregation, DNA damage, and replication, that are post-transcriptionally regulated during the mammalian cell cycle. Moreover, we show that combining the approaches better predicts steady state changes in protein abundance. The results reported here support the conjecture that for gaining a full post-transcriptional regulation picture one should integrate the two approaches.


Asunto(s)
Ciclo Celular/genética , Biosíntesis de Proteínas/genética , Proteómica , Ribosomas/genética , División Celular/genética , Fase G1/genética , Regulación del Desarrollo de la Expresión Génica/genética , Células HeLa , Humanos , Transducción de Señal
15.
Nat Protoc ; 9(4): 751-60, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24603934

RESUMEN

Regulation of mRNA translation has a pivotal role in modulating protein levels, and the genome-wide identification of proteins synthesized at a given time is indispensable to our understanding of gene expression. This protocol describes the mass-spectrometric analysis of newly synthesized proteins from cultured cells or whole tissues by using a biotinylated derivative of puromycin, which becomes incorporated into nascent polypeptide chains by ribosome catalysis. In this method, termed puromycin-associated nascent chain proteomics (PUNCH-P), intact ribosome-nascent chain complexes are first recovered from cells by ultracentrifugation, followed by biotin-puromycin labeling of newly synthesized proteins, streptavidin affinity purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Unlike methods that require in vivo labeling, the sensitivity and coverage of PUNCH-P depend only on the amount of starting material and not on the duration of labeling, thus enabling the measurement of rapid fluctuations in protein synthesis. The protocol requires 3 d for sample preparation and analysis.


Asunto(s)
Biosíntesis de Proteínas/genética , Proteómica/métodos , Puromicina/química , Espectrometría de Masas en Tándem/métodos , Biotina/química , Células Cultivadas , Cromatografía Liquida , Ribosomas/genética , Ultracentrifugación
16.
Translation (Austin) ; 1(2): e27516, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-26824027

RESUMEN

Regulation of mRNA translation is a major modulator of gene expression, allowing cells to fine tune protein levels during growth and differentiation and in response to physiological signals and environmental changes. Mass-spectrometry and RNA-sequencing methods now enable global profiling of the translatome, but these still involve significant analytical and economical limitations. We developed a novel system-wide proteomic approach for direct monitoring of translation, termed PUromycin-associated Nascent CHain Proteomics (PUNCH-P), which is based on the recovery of ribosome-nascent chain complexes from cells or tissues followed by incorporation of biotinylated puromycin into newly-synthesized proteins. Biotinylated proteins are then purified by streptavidin and analyzed by mass-spectrometry. Here we present an overview of PUNCH-P, describe other methodologies for global translatome profiling (pSILAC, BONCAT, TRAP/Ribo-tag, Ribo-seq) and provide conceptual comparisons between these methods. We also show how PUNCH-P data can be combined with mRNA measurements to determine relative translation efficiency for specific mRNAs.

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