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1.
Mol Cell ; 81(7): 1372-1383, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33713598

RESUMO

Eukaryotic cells integrate multiple quality control (QC) responses during protein synthesis in the cytoplasm. These QC responses are signaled by slow or stalled elongating ribosomes. Depending on the nature of the delay, the signal may lead to translational repression, messenger RNA decay, ribosome rescue, and/or nascent protein degradation. Here, we discuss how the structure and composition of an elongating ribosome in a troubled state determine the downstream quality control pathway(s) that ensue. We highlight the intersecting pathways involved in RNA decay and the crosstalk that occurs between RNA decay and ribosome rescue.


Assuntos
Células Eucarióticas/metabolismo , Biossíntese de Proteínas/fisiologia , Estabilidade de RNA/fisiologia , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Animais , Humanos
2.
Nat Commun ; 12(1): 1778, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33741954

RESUMO

Memory reconsolidation, the process by which memories are again stabilized after being reactivated, has strengthened the idea that memory stabilization is a highly plastic process. To date, the molecular and cellular bases of reconsolidation have been extensively investigated particularly within the hippocampus. However, the role of adult neurogenesis in memory reconsolidation is unclear. Here, we combined functional imaging, retroviral and chemogenetic approaches in rats to tag and manipulate different populations of rat adult-born neurons. We find that both mature and immature adult-born neurons are activated by remote memory retrieval. However, only specific silencing of the adult-born neurons immature during learning impairs remote memory retrieval-induced reconsolidation. Hence, our findings show that adult-born neurons immature during learning are required for the maintenance and update of remote memory reconsolidation.


Assuntos
Aprendizagem/fisiologia , Consolidação da Memória/fisiologia , Memória de Longo Prazo/fisiologia , Neurônios/fisiologia , Animais , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Hipocampo/fisiologia , Masculino , Aprendizagem em Labirinto/fisiologia , Microscopia Confocal , Neurônios/metabolismo , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/fisiologia , Ratos Sprague-Dawley , Fatores de Tempo
3.
Nat Commun ; 12(1): 1547, 2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33707436

RESUMO

Hypertension, exercise, and pregnancy are common triggers of cardiac remodeling, which occurs primarily through the hypertrophy of individual cardiomyocytes. During hypertrophy, stress-induced signal transduction increases cardiomyocyte transcription and translation, which promotes the addition of new contractile units through poorly understood mechanisms. The cardiomyocyte microtubule network is also implicated in hypertrophy, but via an unknown role. Here, we show that microtubules are indispensable for cardiac growth via spatiotemporal control of the translational machinery. We find that the microtubule motor Kinesin-1 distributes mRNAs and ribosomes along microtubule tracks to discrete domains within the cardiomyocyte. Upon hypertrophic stimulation, microtubules redistribute mRNAs and new protein synthesis to sites of growth at the cell periphery. If the microtubule network is disrupted, mRNAs and ribosomes collapse around the nucleus, which results in mislocalized protein synthesis, the rapid degradation of new proteins, and a failure of growth, despite normally increased translation rates. Together, these data indicate that mRNAs and ribosomes are actively transported to specific sites to facilitate local translation and assembly of contractile units, and suggest that properly localized translation - and not simply translation rate - is a critical determinant of cardiac hypertrophy. In this work, we find that microtubule based-transport is essential to couple augmented transcription and translation to productive cardiomyocyte growth during cardiac stress.


Assuntos
Cardiomegalia/patologia , Microtúbulos/metabolismo , Miócitos Cardíacos/patologia , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Animais , Remodelamento Atrial/fisiologia , Transporte Biológico/fisiologia , Células Cultivadas , Humanos , Cinesina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Transdução de Sinais/fisiologia , Remodelação Ventricular/fisiologia
4.
Nat Chem Biol ; 17(4): 421-427, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542534

RESUMO

Coupled transcription and translation processes in bacteria cause indiscriminate translation of intact and truncated messenger RNAs, inevitably generating nonfunctional polypeptides. Here, we devised a synthetic protein quality control (ProQC) system that enables translation only when both ends of mRNAs are present and followed by circularization based on sequence-specific RNA-RNA hybridization. We demonstrate that the ProQC system dramatically improved the fraction of full-length proteins among all synthesized polypeptides by selectively translating intact mRNA and reducing abortive translation. As a result, full-length protein synthesis increased up to 2.5-fold without changing the transcription or translation efficiency. Furthermore, we applied the ProQC system for 3-hydroxypropionic acid, violacein and lycopene production by ensuring full-length expression of enzymes in biosynthetic pathways, resulting in 1.6- to 2.3-fold greater biochemical production. We believe that our ProQC system can be universally applied to improve not only the quality of recombinant protein production but also efficiencies of metabolic pathways.


Assuntos
Engenharia Genética/métodos , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/fisiologia , Bactérias/genética , Sequência de Bases/genética , Escherichia coli/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Controle de Qualidade , RNA Mensageiro/química , RNA Mensageiro/metabolismo
5.
Obes Rev ; 22(4): e13221, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33569904

RESUMO

Obesity and obesogenic comorbidities have been associated with COVID-19 susceptibility and mortality. However, the mechanism of such correlations requires an in-depth understanding. Overnutrition/excess serum amino acid profile during obesity has been linked with inflammation and reprogramming of translational machinery through hyperactivation of amino acid sensor mammalian target of rapamycin (mTOR), which is exploited by SARS-CoV-2 for its replication. Conversely, we have shown that the activation of general control nonderepressible 2 (GCN2)-dependent amino acid starvation sensing pathway suppresses intestinal inflammation by inhibiting the production of reactive oxygen species (ROS) and interleukin-1 beta (IL-1ß). While activation of GCN2 has shown to mitigate susceptibility to dengue infection, GCN2 deficiency increases viremia and inflammation-associated pathologies. These findings reveal that the amino acid sensing pathway plays a significant role in controlling inflammation and viral infections. The current fact is that obesity/excess amino acids/mTOR activation aggravates COVID-19, and it might be possible that activation of amino acid starvation sensor GCN2 has an opposite effect. This article focuses on the amino acid sensing pathways through which host cells sense the availability of amino acids and reprogram the host translation machinery to mount an effective antiviral response. Besides, how SARS-CoV-2 hijack and exploit amino acid sensing pathway for its replication and pathogenesis is also discussed.


Assuntos
Aminoácidos/metabolismo , N-Acetilexosaminiltransferases/fisiologia , Obesidade/epidemiologia , /metabolismo , Comorbidade , Humanos , Inflamação , Obesidade/fisiopatologia , Biossíntese de Proteínas/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Replicação Viral/fisiologia
6.
Nat Commun ; 12(1): 479, 2021 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-33473112

RESUMO

As organisms develop, individual cells generate mitochondria to fulfill physiological requirements. However, it remains unknown how mitochondrial network expansion is scaled to cell growth. The mitochondrial unfolded protein response (UPRmt) is a signaling pathway mediated by the transcription factor ATFS-1 which harbors a mitochondrial targeting sequence (MTS). Here, using the model organism Caenorhabditis elegans we demonstrate that ATFS-1 mediates an adaptable mitochondrial network expansion program that is active throughout normal development. Mitochondrial network expansion requires the relatively inefficient MTS in ATFS-1, which allows the transcription factor to be responsive to parameters that impact protein import capacity of the mitochondrial network. Increasing the strength of the ATFS-1 MTS impairs UPRmt activity by increasing accumulation within mitochondria. Manipulations of TORC1 activity increase or decrease ATFS-1 activity in a manner that correlates with protein synthesis. Lastly, expression of mitochondrial-targeted GFP is sufficient to expand the muscle cell mitochondrial network in an ATFS-1-dependent manner. We propose that mitochondrial network expansion during development is an emergent property of the synthesis of highly expressed mitochondrial proteins that exclude ATFS-1 from mitochondrial import, causing UPRmt activation.


Assuntos
Proteínas de Caenorhabditis elegans/biossíntese , Caenorhabditis elegans/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Biossíntese de Proteínas/fisiologia , Animais , Caenorhabditis elegans/genética , Metabolismo Energético , Regulação da Expressão Gênica , Chaperonas Moleculares , Transporte Proteico , Transdução de Sinais , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas
7.
Nat Struct Mol Biol ; 28(1): 103-117, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33398172

RESUMO

Although polycomb repressive complex 2 (PRC2) is now recognized as an RNA-binding complex, the full range of binding motifs and why PRC2-RNA complexes often associate with active genes have not been elucidated. Here, we identify high-affinity RNA motifs whose mutations weaken PRC2 binding and attenuate its repressive function in mouse embryonic stem cells. Interactions occur at promoter-proximal regions and frequently coincide with pausing of RNA polymerase II (POL-II). Surprisingly, while PRC2-associated nascent transcripts are highly expressed, ablating PRC2 further upregulates expression via loss of pausing and enhanced transcription elongation. Thus, PRC2-nascent RNA complexes operate as rheostats to fine-tune transcription by regulating transitions between pausing and elongation, explaining why PRC2-RNA complexes frequently occur within active genes. Nascent RNA also targets PRC2 in cis and downregulates neighboring genes. We propose a unifying model in which RNA specifically recruits PRC2 to repress genes through POL-II pausing and, more classically, trimethylation of histone H3 at Lys27.


Assuntos
Regulação da Expressão Gênica/genética , Complexo Repressor Polycomb 2/metabolismo , Biossíntese de Proteínas/fisiologia , RNA Polimerase II/metabolismo , RNA/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Histonas/metabolismo , Metilação , Camundongos , Motivos de Nucleotídeos/genética , Regiões Promotoras Genéticas/genética , Biossíntese de Proteínas/genética , RNA/genética , Transcrição Genética/genética , Ativação Transcricional/genética
8.
Cancer Res ; 81(3): 517-524, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33479028

RESUMO

Deregulation of the mRNA translational process has been observed during tumorigenesis. However, recent findings have shown that deregulation of translation also contributes specifically to cancer cell spread. During metastasis, cancer cells undergo changes in cellular state, permitting the acquisition of features necessary for cell survival, dissemination, and outgrowth. In addition, metastatic cells respond to external cues, allowing for their persistence under significant cellular and microenvironmental stresses. Recent work has revealed the importance of mRNA translation to these dynamic changes, including regulation of cell states through epithelial-to-mesenchymal transition and tumor dormancy and as a response to external stresses such as hypoxia and immune surveillance. In this review, we focus on examples of altered translation underlying these phenotypic changes and responses to external cues and explore how they contribute to metastatic progression. We also highlight the therapeutic opportunities presented by aberrant mRNA translation, suggesting novel ways to target metastatic tumor cells.


Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Metástase Neoplásica/genética , Biossíntese de Proteínas/fisiologia , Carcinogênese/metabolismo , Movimento Celular , Sobrevivência Celular/fisiologia , Humanos , Metástase Neoplásica/patologia , Metástase Neoplásica/terapia , Proteínas de Neoplasias/biossíntese , Neoplasias/terapia , Neovascularização Patológica/etiologia , Fenótipo , Serina-Treonina Quinases TOR/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Evasão Tumoral/fisiologia , Hipóxia Tumoral/fisiologia , Microambiente Tumoral/fisiologia
9.
Mol Pharmacol ; 99(1): 1-16, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33130557

RESUMO

Aberrant activation of Wnt/ß-catenin axis occurs in several gastrointestinal malignancies due to inactivating mutations of adenomatous polyposis coli (in colorectal cancer) or activating mutations of ß-catenin itself [in hepatocellular carcinoma (HCC)]. These lead to ß-catenin stabilization, increase in ß-catenin/T-cell factor (TCF)-mediated transcriptional activation, and target gene expression, many of which are involved in tumor progression. While studying pharmaceutical agents that can target ß-catenin in cancer cells, we observed that the plant compound berberine (BBR), a potent activator of AMP-activated protein kinase (AMPK), can reduce ß-catenin expression and downstream signaling in HCC cells in a dose-dependent manner. More in-depth analyses to understand the mechanism revealed that BBR-induced reduction of ß-catenin occurs independently of AMPK activation and does not involve transcriptional or post-translational mechanisms. Pretreatment with protein synthesis inhibitor cycloheximide antagonized BBR-induced ß-catenin reduction, suggesting that BBR affects ß-catenin translation. BBR treatment also antagonized mammalian target of rapamycin (mTOR) activity and was associated with increased recruitment of eukaryotic translation initiation factor 4E-binding protein (4E-BP) 1 in the translational complex, which was revealed by 7-methyl-cap-binding assays, suggesting inhibition of cap-dependent translation. Interestingly, knocking down 4E-BP1 and 4E-BP2 significantly attenuated BBR-induced reduction of ß-catenin levels and expression of its downstream target genes. Moreover, cells with 4E-BP knockdown were resistant to BBR-induced cell death and were resensitized to BBR after pharmacological inhibition of ß-catenin. Our findings indicate that BBR antagonizes ß-catenin pathway by inhibiting ß-catenin translation and mTOR activity and thereby reduces HCC cell survival. These also suggest that BBR could be used for targeting HCCs that express mutated/activated ß-catenin variants that are currently undruggable. SIGNIFICANCE STATEMENT: ß-catenin signaling is aberrantly activated in different gastrointestinal cancers, including hepatocellular carcinoma, which is currently undruggable. In this study we describe a novel mechanism of targeting ß-catenin translation via utilizing a plant compound, berberine. Our findings provide a new avenue of targeting ß-catenin axis in cancer, which can be utilized toward the designing of effective therapeutic strategies to combat ß-catenin-dependent cancers.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Berberina/farmacologia , Carcinoma Hepatocelular/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Neoplasias Hepáticas/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinoma Hepatocelular/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Fatores de Iniciação em Eucariotos/antagonistas & inibidores , Fatores de Iniciação em Eucariotos/genética , Células HEK293 , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/fisiologia , beta Catenina/antagonistas & inibidores , beta Catenina/genética
10.
Nucleic Acids Res ; 49(1): 458-478, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33332560

RESUMO

The mammalian target of rapamycin (mTOR) is a critical regulator of cell growth, integrating multiple signalling cues and pathways. Key among the downstream activities of mTOR is the control of the protein synthesis machinery. This is achieved, in part, via the co-ordinated regulation of mRNAs that contain a terminal oligopyrimidine tract (TOP) at their 5'ends, although the mechanisms by which this occurs downstream of mTOR signalling are still unclear. We used RNA-binding protein (RBP) capture to identify changes in the protein-RNA interaction landscape following mTOR inhibition. Upon mTOR inhibition, the binding of LARP1 to a number of mRNAs, including TOP-containing mRNAs, increased. Importantly, non-TOP-containing mRNAs bound by LARP1 are in a translationally-repressed state, even under control conditions. The mRNA interactome of the LARP1-associated protein PABPC1 was found to have a high degree of overlap with that of LARP1 and our data show that PABPC1 is required for the association of LARP1 with its specific mRNA targets. Finally, we demonstrate that mRNAs, including those encoding proteins critical for cell growth and survival, are translationally repressed when bound by both LARP1 and PABPC1.


Assuntos
Autoantígenos/fisiologia , Proteína I de Ligação a Poli(A)/fisiologia , Polirribossomos/metabolismo , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/metabolismo , Ribonucleoproteínas/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Regiões 5' não Traduzidas/genética , Autoantígenos/genética , Regulação da Expressão Gênica , Genes Reporter , Células HeLa , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 2 de Rapamicina/antagonistas & inibidores , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Naftiridinas/farmacologia , Mutação Puntual , Biossíntese de Proteínas/genética , Interferência de RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas/genética
11.
Methods Mol Biol ; 2192: 243-268, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33230778

RESUMO

Cryo-electron tomography (cryo-ET) enables the three-dimensional (3D) visualization of macromolecular complexes in their native environment (in situ). The ability to visualize macromolecules in situ is in particular advantageous for complex, membrane-associated processes, such as mitochondrial translation. Mitochondrial translation occurs almost exclusively associated with the inner mitochondrial membrane, giving rise to the mitochondrial DNA-encoded subunits of oxidative phosphorylation machinery. In cryo-ET, the 3D volume is reconstructed from a set of 2D projections of a frozen-hydrated specimen, which is sequentially tilted and imaged at different angles in a transmission electron microscope. In combination with subtomogram analysis, cryo-ET enables the structure determination of macromolecular complexes and their 3D organization. In this chapter, we summarize all steps required for structural characterization of mitochondrial ribosomes in situ, ranging from data acquisition to tomogram reconstruction and subtomogram analysis.


Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Mitocôndrias/metabolismo , Biossíntese de Proteínas/fisiologia , Células HEK293 , Humanos , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Substâncias Macromoleculares/química , Software
12.
Int J Mol Sci ; 21(24)2020 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-33322589

RESUMO

The fidelity of protein synthesis, a process shaped by several mechanisms involving specialized ribosome regions and external factors, ensures the precise reading of sense and stop codons. However, premature termination codons (PTCs) arising from mutations may, at low frequency, be misrecognized and result in PTC suppression, named ribosome readthrough, with production of full-length proteins through the insertion of a subset of amino acids. Since some drugs have been identified as readthrough inducers, this fidelity drawback has been explored as a therapeutic approach in several models of human diseases caused by nonsense mutations. Here, we focus on the mechanisms driving translation in normal and aberrant conditions, the potential fates of mRNA in the presence of a PTC, as well as on the results obtained in the research of efficient readthrough-inducing compounds. In particular, we describe the molecular determinants shaping the outcome of readthrough, namely the nucleotide and protein context, with the latter being pivotal to produce functional full-length proteins. Through the interpretation of experimental and mechanistic findings, mainly obtained in lysosomal and coagulation disorders, we also propose a scenario of potential readthrough-favorable features to achieve relevant rescue profiles, representing the main issue for the potential translatability of readthrough as a therapeutic strategy.


Assuntos
Códon sem Sentido/genética , Códon de Terminação/genética , Animais , Humanos , Mutação/genética , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/fisiologia , Ribossomos/metabolismo
13.
Elife ; 92020 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-33357378

RESUMO

Key enzymatic processes use the nonequilibrium error correction mechanism called kinetic proofreading to enhance their specificity. The applicability of traditional proofreading schemes, however, is limited because they typically require dedicated structural features in the enzyme, such as a nucleotide hydrolysis site or multiple intermediate conformations. Here, we explore an alternative conceptual mechanism that achieves error correction by having substrate binding and subsequent product formation occur at distinct physical locations. The time taken by the enzyme-substrate complex to diffuse from one location to another is leveraged to discard wrong substrates. This mechanism does not have the typical structural requirements, making it easier to overlook in experiments. We discuss how the length scales of molecular gradients dictate proofreading performance, and quantify the limitations imposed by realistic diffusion and reaction rates. Our work broadens the applicability of kinetic proofreading and sets the stage for studying spatial gradients as a possible route to specificity.


Assuntos
Replicação do DNA/fisiologia , Cinética , Biossíntese de Proteínas/fisiologia , Especificidade por Substrato/fisiologia , Fenômenos Biofísicos , Hidrólise , Modelos Biológicos
14.
PLoS One ; 15(12): e0236850, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33315868

RESUMO

Ribosomes are the sophisticated machinery that is responsible for protein synthesis in a cell. Recently, quantitative mass spectrometry (qMS) have been successfully applied for understanding the dynamics of protein complexes. Here, we developed a highly specific and reproducible method to quantify all ribosomal proteins (r-proteins) by combining selected reaction monitoring (SRM) and isotope labeling. We optimized the SRM methods using purified ribosomes and Escherichia coli lysates and verified this approach as detecting 41 of the 54 r-proteins separately synthesized in E. coli S30 extracts. The SRM methods will enable us to utilize qMS as a highly specific analytical tool in the research of E. coli ribosomes, and this methodology have potential to accelerate the understanding of ribosome biogenesis, function, and the development of engineered ribosomes with additional functions.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Marcação por Isótopo/métodos , Espectrometria de Massas/métodos , Biossíntese de Proteínas/fisiologia , RNA Ribossômico/metabolismo , Subunidades Ribossômicas Menores de Bactérias/metabolismo
15.
Nat Commun ; 11(1): 5052, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-33028817

RESUMO

The mechanism and function of autophagy as a highly-conserved bulk degradation pathway are well studied, but the physiological role of autophagy remains poorly understood. We show that autophagy is involved in the adaptation of Saccharomyces cerevisiae to respiratory growth through its recycling of serine. On respiratory media, growth onset, mitochondrial initiator tRNA modification and mitochondrial protein expression are delayed in autophagy defective cells, suggesting that mitochondrial one-carbon metabolism is perturbed in these cells. The supplementation of serine, which is a key one-carbon metabolite, is able to restore mitochondrial protein expression and alleviate delayed respiratory growth. These results indicate that autophagy-derived serine feeds into mitochondrial one-carbon metabolism, supporting the initiation of mitochondrial protein synthesis and allowing rapid adaptation to respiratory growth.


Assuntos
Adaptação Fisiológica , Autofagia/fisiologia , Proteínas Mitocondriais/biossíntese , Saccharomyces cerevisiae/fisiologia , Carbono/metabolismo , Respiração Celular/fisiologia , Mitocôndrias/metabolismo , Biossíntese de Proteínas/fisiologia , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/metabolismo
16.
PLoS One ; 15(10): e0239700, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33017414

RESUMO

In the past two decades, research into the biochemical, biophysical and structural properties of the ribosome have revealed many different steps of protein translation. Nevertheless, a complete understanding of how they lead to a rapid and accurate protein synthesis still remains a challenge. Here we consider a coarse network analysis in the bacterial ribosome formed by the connectivity between ribosomal (r) proteins and RNAs at different stages in the elongation cycle. The ribosomal networks are found to be dis-assortative and small world, implying that the structure allows for an efficient exchange of information between distant locations. An analysis of centrality shows that the second and fifth domains of 23S rRNA are the most important elements in all of the networks. Ribosomal protein hubs connect to much fewer nodes but are shown to provide important connectivity within the network (high closeness centrality). A modularity analysis reveals some of the different functional communities, indicating some known and some new possible communication pathways Our mathematical results confirm important communication pathways that have been discussed in previous research, thus verifying the use of this technique for representing the ribosome, and also reveal new insights into the collective function of ribosomal elements.


Assuntos
Bactérias/genética , Redes Reguladoras de Genes/genética , Ribossomos/genética , Bactérias/metabolismo , Biologia Computacional/métodos , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/fisiologia , RNA Ribossômico 23S/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Elongação da Transcrição Genética/fisiologia
17.
Proc Natl Acad Sci U S A ; 117(35): 21775-21784, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817480

RESUMO

The D1 reaction center protein of photosystem II (PSII) is subject to light-induced damage. Degradation of damaged D1 and its replacement by nascent D1 are at the heart of a PSII repair cycle, without which photosynthesis is inhibited. In mature plant chloroplasts, light stimulates the recruitment of ribosomes specifically to psbA mRNA to provide nascent D1 for PSII repair and also triggers a global increase in translation elongation rate. The light-induced signals that initiate these responses are unclear. We present action spectrum and genetic data indicating that the light-induced recruitment of ribosomes to psbA mRNA is triggered by D1 photodamage, whereas the global stimulation of translation elongation is triggered by photosynthetic electron transport. Furthermore, mutants lacking HCF136, which mediates an early step in D1 assembly, exhibit constitutively high psbA ribosome occupancy in the dark and differ in this way from mutants lacking PSII for other reasons. These results, together with the recent elucidation of a thylakoid membrane complex that functions in PSII assembly, PSII repair, and psbA translation, suggest an autoregulatory mechanism in which the light-induced degradation of D1 relieves repressive interactions between D1 and translational activators in the complex. We suggest that the presence of D1 in this complex coordinates D1 synthesis with the need for nascent D1 during both PSII biogenesis and PSII repair in plant chloroplasts.


Assuntos
Proteínas de Arabidopsis/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Ligação à Clorofila/metabolismo , Cloroplastos/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Luz , Proteínas de Membrana/metabolismo , Fotossíntese , Complexo de Proteína do Fotossistema II/genética , Plantas/genética , Biossíntese de Proteínas/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Transcrição Genética , Zea mays/genética , Zea mays/metabolismo
18.
Nat Commun ; 11(1): 4013, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32782250

RESUMO

Antibiotics that interfere with translation, when combined, interact in diverse and difficult-to-predict ways. Here, we explain these interactions by "translation bottlenecks": points in the translation cycle where antibiotics block ribosomal progression. To elucidate the underlying mechanisms of drug interactions between translation inhibitors, we generate translation bottlenecks genetically using inducible control of translation factors that regulate well-defined translation cycle steps. These perturbations accurately mimic antibiotic action and drug interactions, supporting that the interplay of different translation bottlenecks causes these interactions. We further show that growth laws, combined with drug uptake and binding kinetics, enable the direct prediction of a large fraction of observed interactions, yet fail to predict suppression. However, varying two translation bottlenecks simultaneously supports that dense traffic of ribosomes and competition for translation factors account for the previously unexplained suppression. These results highlight the importance of "continuous epistasis" in bacterial physiology.


Assuntos
Antibacterianos/farmacologia , Modelos Teóricos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Interações Medicamentosas , Epistasia Genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Biossíntese de Proteínas/fisiologia , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo
19.
Proc Natl Acad Sci U S A ; 117(27): 15581-15590, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32576685

RESUMO

Protein synthesis represents a major metabolic activity of the cell. However, how it is affected by aging and how this in turn impacts cell function remains largely unexplored. To address this question, herein we characterized age-related changes in both the transcriptome and translatome of mouse tissues over the entire life span. We showed that the transcriptome changes govern those in the translatome and are associated with altered expression of genes involved in inflammation, extracellular matrix, and lipid metabolism. We also identified genes that may serve as candidate biomarkers of aging. At the translational level, we uncovered sustained down-regulation of a set of 5'-terminal oligopyrimidine (5'-TOP) transcripts encoding protein synthesis and ribosome biogenesis machinery and regulated by the mTOR pathway. For many of them, ribosome occupancy dropped twofold or even more. Moreover, with age, ribosome coverage gradually decreased in the vicinity of start codons and increased near stop codons, revealing complex age-related changes in the translation process. Taken together, our results reveal systematic and multidimensional deregulation of protein synthesis, showing how this major cellular process declines with age.


Assuntos
Envelhecimento/fisiologia , Regulação da Expressão Gênica/fisiologia , Biossíntese de Proteínas/fisiologia , Ribossomos/metabolismo , Animais , Códon de Iniciação/metabolismo , Biologia Computacional , Masculino , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA-Seq , Ribossomos/genética , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Transcriptoma/fisiologia
20.
Nat Commun ; 11(1): 3146, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561718

RESUMO

Phosphorylation of intrinsically disordered eIF4E binding proteins (4E-BPs) regulates cap-dependent translation by weakening their ability to compete with eIF4G for eIF4E binding within the translation initiation complex. We previously showed that phosphorylation of T37 and T46 in 4E-BP2 induces folding of a four-stranded beta-fold domain, partially sequestering the canonical eIF4E-binding helix. The C-terminal intrinsically disordered region (C-IDR), remaining disordered after phosphorylation, contains the secondary eIF4E-binding site and three other phospho-sites, whose mechanisms in inhibiting binding are not understood. Here we report that the domain is non-cooperatively folded, with exchange between beta strands and helical conformations. C-IDR phosphorylation shifts the conformational equilibrium, controlling access to eIF4E binding sites. The hairpin turns formed by pT37/pT46 are remarkably stable and function as transplantable units for phospho-regulation of stability. These results demonstrate how non-cooperative folding and conformational exchange leads to graded inhibition of 4E-BP2:eIF4E binding, shifting 4E-BP2 into an eIF4E binding-incompatible conformation and regulating translation initiation.


Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Biossíntese de Proteínas/fisiologia , Capuzes de RNA/metabolismo , Biologia Computacional , Fator de Iniciação 4E em Eucariotos/genética , Proteínas Intrinsicamente Desordenadas/genética , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Fosforilação/fisiologia , Ligação Proteica/genética , Conformação Proteica em alfa-Hélice/genética , Conformação Proteica em Folha beta/genética , Dobramento de Proteína , Processamento de Proteína Pós-Traducional/fisiologia
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