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1.
Nat Commun ; 12(1): 1799, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33741965

RESUMO

Bacterial ribosome rescue pathways that remove ribosomes stalled on mRNAs during translation have been proposed as novel antibiotic targets because they are essential in bacteria and are not conserved in humans. We previously reported the discovery of a family of acylaminooxadiazoles that selectively inhibit trans-translation, the main ribosome rescue pathway in bacteria. Here, we report optimization of the pharmacokinetic and antibiotic properties of the acylaminooxadiazoles, producing MBX-4132, which clears multiple-drug resistant Neisseria gonorrhoeae infection in mice after a single oral dose. Single particle cryogenic-EM studies of non-stop ribosomes show that acylaminooxadiazoles bind to a unique site near the peptidyl-transfer center and significantly alter the conformation of ribosomal protein bL27, suggesting a novel mechanism for specific inhibition of trans-translation by these molecules. These results show that trans-translation is a viable therapeutic target and reveal a new conformation within the bacterial ribosome that may be critical for ribosome rescue pathways.


Assuntos
Neisseria gonorrhoeae/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Ribossomos/efeitos dos fármacos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Células CACO-2 , Feminino , Gonorreia/microbiologia , Gonorreia/prevenção & controle , Humanos , Camundongos , Neisseria gonorrhoeae/genética , Biossíntese de Proteínas/genética , Inibidores da Síntese de Proteínas/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
2.
Nat Commun ; 12(1): 1859, 2021 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33767140

RESUMO

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoproteins initiates co-transcriptionally and requires the action of the assembly machinery including the Hsp90/R2TP complex, the Rsa1p:Hit1p heterodimer and the Bcd1 protein. We present genetic interactions between the Rsa1p-encoding gene and genes involved in chromatin organization including RTT106 that codes for the H3-H4 histone chaperone Rtt106p controlling H3K56ac deposition. We show that Bcd1p binds Rtt106p and controls its transcription-dependent recruitment by reducing its association with RNA polymerase II, modulating H3K56ac levels at gene body. We reveal the 3D structures of the free and Rtt106p-bound forms of Bcd1p using nuclear magnetic resonance and X-ray crystallography. The interaction is also studied by a combination of biophysical and proteomic techniques. Bcd1p interacts with a region that is distinct from the interaction interface between the histone chaperone and histone H3. Our results are evidence for a protein interaction interface for Rtt106p that controls its transcription-associated activity.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Chaperonas Moleculares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ativação Transcricional/fisiologia , Proliferação de Células/fisiologia , Cromatina/genética , Cristalografia por Raios X , Histonas/metabolismo , Ressonância Magnética Nuclear Biomolecular , RNA Polimerase II/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transcrição Genética/genética
3.
Medicine (Baltimore) ; 100(8): e24898, 2021 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-33663122

RESUMO

ABSTRACT: Ovarian cancer (OC) is the leading cause of gynecological cancer deaths. Extraordinary histologic and genetic heterogeneity presents as great hurdle to OC's diagnosis and treatment. MRPS12 (Mitochondrial Ribosomal Protein S12), encoding a 28S subunit protein, controls the decoding fidelity and susceptibility to aminoglycoside antibiotics. Our study aims to investigate the clinical significance and potential mechanism of MRPS12 in OC.Oncomine, Tumor Immune Estimation Resource database (TIMER), and GEPIA databases were utilized to explore the expression level of MRPS12 in OC and normal tissues. Kaplan-Meier plotter was used to evaluate the influence of MRPS12 expression on OC patients' survival. The potential biologic function and immune infiltration of MRPS12 in OC were analyzed by GSEA (Gene set enrichment analysis) and TIMER database, respectively.MRPS12 was significantly highly expressed in OC (P < .05) compared with normal ovarian tissues. Its overexpression was also significantly related with poor overall survival in advanced FIGO stage (III+IV) patients, in serous OC and in those patients with TP53 mutation (P < .05). GSEA showed that HALLMARK_G2M_CHECKPOINT, BIOCARTA_CELLCYCLE_PATHWAY, HALLMARK_PI3K_AKT_MTOR_SIGNALING, BIOCARTA_P53_PATHWAY were significantly enriched in high-MRPS12-expression phenotype. MRPS12 expression was positively correlated with the infiltration of macrophages and neutrophils in OC.These results reveal that MRPS12 could function as a potential oncogene and serve as a promising prognostic candidate in OC.


Assuntos
Carcinoma Epitelial do Ovário/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Mitocondriais/metabolismo , Neoplasias Ovarianas/genética , Proteínas Ribossômicas/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Epitelial do Ovário/mortalidade , Feminino , Humanos , Estimativa de Kaplan-Meier , Oncogenes , Neoplasias Ovarianas/mortalidade
4.
Nucleic Acids Res ; 49(5): 2894-2915, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33619526

RESUMO

Trans-acting regulatory RNAs have the capacity to base pair with more mRNAs than generally detected under defined conditions, raising the possibility that sRNA target specificities vary depending on the specific metabolic or environmental conditions. In Sinorhizobium meliloti, the sRNA rnTrpL is derived from a tryptophan (Trp) transcription attenuator located upstream of the Trp biosynthesis gene trpE(G). The sRNA rnTrpL contains a small ORF, trpL, encoding the 14-aa leader peptide peTrpL. If Trp is available, efficient trpL translation causes transcription termination and liberation of rnTrpL, which subsequently acts to downregulate the trpDC operon, while peTrpL is known to have a Trp-independent role in posttranscriptional regulation of antibiotic resistance mechanisms. Here, we show that tetracycline (Tc) causes rnTrpL accumulation independently of Trp availability. In the presence of Tc, rnTrpL and peTrpL act collectively to destabilize rplUrpmA mRNA encoding ribosomal proteins L21 and L27. The three molecules, rnTrpL, peTrpL, and rplUrpmA mRNA, form an antibiotic-dependent ribonucleoprotein complex (ARNP). In vitro reconstitution of this ARNP in the presence of competing trpD and rplU transcripts revealed that peTrpL and Tc cause a shift of rnTrpL specificity towards rplU, suggesting that sRNA target prioritization may be readjusted in response to changing environmental conditions.


Assuntos
Antibacterianos/farmacologia , Peptídeos/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/metabolismo , Sinorhizobium meliloti/genética , Tetraciclina/farmacologia , Pareamento de Bases , Regulação Bacteriana da Expressão Gênica , Peptídeos/química , RNA Antissenso/metabolismo , RNA Mensageiro/química , Pequeno RNA não Traduzido/química , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Sinorhizobium meliloti/efeitos dos fármacos
5.
Nat Commun ; 12(1): 782, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542241

RESUMO

The guided entry of tail-anchored proteins (GET) pathway assists in the posttranslational delivery of tail-anchored proteins, containing a single C-terminal transmembrane domain, to the ER. Here we uncover how the yeast GET pathway component Get4/5 facilitates capture of tail-anchored proteins by Sgt2, which interacts with tail-anchors and hands them over to the targeting component Get3. Get4/5 binds directly and with high affinity to ribosomes, positions Sgt2 close to the ribosomal tunnel exit, and facilitates the capture of tail-anchored proteins by Sgt2. The contact sites of Get4/5 on the ribosome overlap with those of SRP, the factor mediating cotranslational ER-targeting. Exposure of internal transmembrane domains at the tunnel exit induces high-affinity ribosome binding of SRP, which in turn prevents ribosome binding of Get4/5. In this way, the position of a transmembrane domain within nascent ER-targeted proteins mediates partitioning into either the GET or SRP pathway directly at the ribosomal tunnel exit.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Adenosina Trifosfatases/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Mutação , Terminação Traducional da Cadeia Peptídica , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Partícula de Reconhecimento de Sinal/metabolismo , Ubiquitina/genética , Ubiquitina/isolamento & purificação
6.
Vet Res ; 52(1): 30, 2021 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-33618766

RESUMO

Host proteins interacting with pathogens are receiving more attention as potential therapeutic targets in molecular medicine. Streptococcus suis serotype 2 (SS2) is an important cause of meningitis in both humans and pigs worldwide. SS2 Enolase (Eno) has previously been identified as a virulence factor with a role in altering blood brain barrier (BBB) integrity, but the host cell membrane receptor of Eno and The mechanism(s) involved are unclear. This study identified that SS2 Eno binds to 40S ribosomal protein SA (RPSA) on the surface of porcine brain microvascular endothelial cells leading to activation of intracellular p38/ERK-eIF4E signalling, which promotes intracellular expression of HSPD1 (heat-shock protein family D member 1), and initiation of host-cell apoptosis, and increased BBB permeability facilitating bacterial invasion. This study reveals novel functions for the host-interactional molecules RPSA and HSPD1 in BBB integrity, and provides insight for new therapeutic strategies in meningitis.


Assuntos
Barreira Hematoencefálica , Células Endoteliais/metabolismo , Fosfopiruvato Hidratase/metabolismo , Proteínas Ribossômicas/metabolismo , Infecções Estreptocócicas/veterinária , Streptococcus suis/metabolismo , Animais , Apoptose , Técnicas de Cocultura , Células Endoteliais/microbiologia , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Camundongos , Ligação Proteica , Sorogrupo , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/patologia , Streptococcus suis/patogenicidade , Suínos , Doenças dos Suínos/microbiologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
7.
Nat Commun ; 12(1): 335, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436550

RESUMO

Previous transcriptomic profiling studies have typically focused on separately analyzing mRNA expression, alternative splicing and alternative polyadenylation differences between cell and tissue types. However, the relative contribution of these three transcriptomic regulatory layers to cell type specification is poorly understood. This question is particularly relevant to neurons, given their extensive heterogeneity associated with brain location, morphology and function. In the present study, we generated profiles for the three regulatory layers from developmentally and regionally distinct subpopulations of neurons from the mouse hippocampus and broader nervous system. Multi-omics factor analyses revealed differing contributions of each transcriptomic layer in the discrimination of neurons based on their stage of development, region, and function. Importantly, profiles of differential alternative splicing and polyadenylation better discriminated specific neuronal subtype populations than gene expression patterns. These results provide evidence for differential relative contributions of coordinated gene regulatory layers in the specification of neuronal subtypes.


Assuntos
Regulação da Expressão Gênica , Neurônios/metabolismo , Transcriptoma/genética , Regiões 3' não Traduzidas/genética , Processamento Alternativo/genética , Animais , Regulação para Baixo/genética , Hipocampo/anatomia & histologia , Hipocampo/citologia , Camundongos , Poliadenilação/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Transcrição Genética , Regulação para Cima/genética
8.
Nat Cell Biol ; 23(2): 136-146, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33495633

RESUMO

Cell competition allows winner cells to eliminate less fit loser cells in tissues. In Minute cell competition, cells with a heterozygous mutation in ribosome genes, such as RpS3+/- cells, are eliminated by wild-type cells. How cells are primed as losers is partially understood and it has been proposed that reduced translation underpins the loser status of ribosome mutant, or Minute, cells. Here, using Drosophila, we show that reduced translation does not cause cell competition. Instead, we identify proteotoxic stress as the underlying cause of the loser status for Minute competition and competition induced by mahjong, an unrelated loser gene. RpS3+/- cells exhibit reduced autophagic and proteasomal flux, accumulate protein aggregates and can be rescued from competition by improving their proteostasis. Conversely, inducing proteotoxic stress is sufficient to turn otherwise wild-type cells into losers. Thus, we propose that tissues may preserve their health through a proteostasis-based mechanism of cell competition and cell selection.


Assuntos
Drosophila melanogaster/citologia , Proteínas/toxicidade , Estresse Fisiológico , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/ultraestrutura , Proteínas de Fluorescência Verde/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Agregados Proteicos , Biossíntese de Proteínas/efeitos dos fármacos , Proteostase/efeitos dos fármacos , Proteínas Ribossômicas/metabolismo , Estresse Fisiológico/efeitos dos fármacos
9.
Proc Natl Acad Sci U S A ; 118(6)2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33479166

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a beta-CoV that recently emerged as a human pathogen and is the causative agent of the COVID-19 pandemic. A molecular framework of how the virus manipulates host cellular machinery to facilitate infection remains unclear. Here, we focus on SARS-CoV-2 NSP1, which is proposed to be a virulence factor that inhibits protein synthesis by directly binding the human ribosome. We demonstrate biochemically that NSP1 inhibits translation of model human and SARS-CoV-2 messenger RNAs (mRNAs). NSP1 specifically binds to the small (40S) ribosomal subunit, which is required for translation inhibition. Using single-molecule fluorescence assays to monitor NSP1-40S subunit binding in real time, we determine that eukaryotic translation initiation factors (eIFs) allosterically modulate the interaction of NSP1 with ribosomal preinitiation complexes in the absence of mRNA. We further elucidate that NSP1 competes with RNA segments downstream of the start codon to bind the 40S subunit and that the protein is unable to associate rapidly with 80S ribosomes assembled on an mRNA. Collectively, our findings support a model where NSP1 proteins from viruses in at least two subgenera of beta-CoVs associate with the open head conformation of the 40S subunit to inhibit an early step of translation, by preventing accommodation of mRNA within the entry channel.


Assuntos
/genética , /virologia , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Proteínas não Estruturais Virais/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Pandemias , Iniciação Traducional da Cadeia Peptídica/genética , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , RNA Viral/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ribossomos/genética , /patogenicidade , Proteínas não Estruturais Virais/genética
10.
Gene ; 776: 145442, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33482283

RESUMO

Ribosome biogenesis requires the concerted activities of three nuclear RNA polymerases, (Pols) I, II, and III, to produce 25S, 18S and 5.8S ribosomal RNA (rRNA), messenger RNA (mRNA) encoding ribosomal proteins, and the 5S rRNA, respectively. The rRNA is processed and modified before being assembled with ribosomal proteins to produce a ribosome. Ribosome biogenesis requires extensive energetic investment by the cell, so it is critical that this process is tightly regulated in accord with cellular growth potential. Previous work revealed that rRNA synthesis in Saccharomyces cerevisiae is repressed prior to the cells shift from active growth (log phase) to limited/static growth (stationary phase). The mechanism(s) by which cells anticipate imminent stationary phase are unknown. In this study, we demonstrate that growing cells produce one or more compounds that accumulate in the growth medium, and that this compound induces repression of rRNA synthesis. When cells encounter this compound, rRNA synthesis is rapidly repressed. We further show that subunits of Pols I and II are degraded during the transition from log to stationary phase growth, but this degradation does not account for the observed repression of rRNA synthesis. Interestingly, repression of rRNA synthesis by spent media requires the nuclear exosome, implying that spent media stimulates rapid rRNA degradation. Together, these data suggest that yeast use quorum sensing to regulate rRNA synthesis in anticipation of high cell density in stationary phase.


Assuntos
Percepção de Quorum/genética , RNA Ribossômico/biossíntese , RNA Ribossômico/genética , Núcleo Celular/metabolismo , Precursores de RNA/genética , Estabilidade de RNA , RNA Ribossômico 5S/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
11.
Nat Commun ; 12(1): 508, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33479206

RESUMO

Thousands of human small and alternative open reading frames (smORFs and alt-ORFs, respectively) have recently been annotated. Many alt-ORFs are co-encoded with canonical proteins in multicistronic configurations, but few of their functions are known. Here, we report the detection of alt-RPL36, a protein co-encoded with human RPL36. Alt-RPL36 partially localizes to the endoplasmic reticulum, where it interacts with TMEM24, which transports the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) precursor phosphatidylinositol from the endoplasmic reticulum to the plasma membrane. Knock-out of alt-RPL36 increases plasma membrane PI(4,5)P2 levels, upregulates PI3K-AKT-mTOR signaling, and increases cell size. Alt-RPL36 contains four phosphoserine residues, point mutations of which abolish interaction with TMEM24 and, consequently, alt-RPL36 effects on PI3K signaling and cell size. These results implicate alt-RPL36 as an upstream regulator of PI3K-AKT-mTOR signaling. More broadly, the RPL36 transcript encodes two sequence-independent polypeptides that co-regulate translation via different molecular mechanisms, expanding our knowledge of multicistronic human gene functions.


Assuntos
Proteínas de Membrana/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Ribossômicas/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico , Membrana Celular/metabolismo , Regulação para Baixo , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/genética , Mutação , Fosfatidilinositol 4,5-Difosfato/metabolismo , Ligação Proteica , Proteínas Ribossômicas/genética
12.
Nat Commun ; 12(1): 599, 2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33500394

RESUMO

The ribosome represents a promising avenue for synthetic biology, but its complexity and essentiality have hindered significant engineering efforts. Heterologous ribosomes, comprising rRNAs and r-proteins derived from different microorganisms, may offer opportunities for novel translational functions. Such heterologous ribosomes have previously been evaluated in E. coli via complementation of a genomic ribosome deficiency, but this method fails to guide the engineering of refractory ribosomes. Here, we implement orthogonal ribosome binding site (RBS):antiRBS pairs, in which engineered ribosomes are directed to researcher-defined transcripts, to inform requirements for heterologous ribosome functionality. We discover that optimized rRNA processing and supplementation with cognate r-proteins enhances heterologous ribosome function for rRNAs derived from organisms with ≥76.1% 16S rRNA identity to E. coli. Additionally, some heterologous ribosomes undergo reduced subunit exchange with E. coli-derived subunits. Cumulatively, this work provides a general framework for heterologous ribosome engineering in living cells.


Assuntos
Escherichia coli/genética , Biossíntese de Proteínas/genética , Proteínas Ribossômicas/genética , Ribossomos/genética , Biologia Sintética/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Filogenia , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Óperon de RNAr/genética
13.
Methods Mol Biol ; 2192: 159-181, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33230773

RESUMO

Human mitochondria contain their own DNA (mtDNA) that encodes 13 proteins all of which are core subunits of oxidative phosphorylation (OXPHOS) complexes. To form functional complexes, these 13 components need to be correctly assembled with approximately 70 nuclear-encoded subunits that are imported following synthesis in the cytosol. How this complicated coordinated translation and assembly is choreographed is still not clear. Methods are being developed to determine whether all members of a particular complex are translated in close proximity, whether protein synthesis is clustered in submitochondrial factories, whether these align with incoming polypeptides, and if there is evidence for co-translational translation that is regulated and limited by the interaction of the incoming proteins with synthesis of their mtDNA-encoded partners. Two methods are described in this chapter to visualize the distribution of mitochondrial ribosomal RNAs in conjunction with newly synthesized mitochondrial proteins. The first combines RNA Fluorescent In Situ Hybridization (FISH) and super-resolution immunocytochemistry to pinpoint mitochondrial ribosomal RNA. The second localizes nascent translation within the mitochondrial network through non-canonical amino acid labeling, click chemistry and fluorescent microscopy.


Assuntos
Química Click/métodos , Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodos , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/metabolismo , RNA Mitocondrial/metabolismo , RNA Ribossômico/metabolismo , Aminoácidos/química , Linhagem Celular Tumoral , DNA Mitocondrial/genética , Humanos , Microscopia de Fluorescência/métodos , Fosforilação Oxidativa , Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo
14.
Methods Mol Biol ; 2192: 197-210, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33230775

RESUMO

Mitochondrial ribosomes (mitoribosomes) are specialized machineries that carry out the synthesis of a limited number of proteins encoded in the mitochondrial genome, including components of the oxidative phosphorylation pathway. They have incorporated several structural features distinguishing them from bacterial and eukaryotic cytosolic counterparts. Our current understanding of the assembly and functioning of mitoribosomes is limited, and recent developments in cryo-EM provide promising directions for detailed investigation. Here we describe methods to purify mitoribosomes from human embryonic kidney cells for cryo-EM studies.


Assuntos
Microscopia Crioeletrônica/métodos , Ribossomos Mitocondriais/química , Ribossomos Mitocondriais/ultraestrutura , Complexo I de Transporte de Elétrons/metabolismo , Processamento Eletrônico de Dados , Genoma Mitocondrial , Células HEK293 , Humanos , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/metabolismo , Fosforilação Oxidativa , Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo
15.
Methods Mol Biol ; 2192: 211-226, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33230776

RESUMO

Mitochondria contain ribosomes (mitoribosomes) specialized in the synthesis of a handful of proteins essential for oxidative phosphorylation. Therefore, mitoribosome integrity and function are essential for the life of eukaryotic cells and lesions that affect them result in devastating human disorders. To broadly analyze the integrity and assembly state of mitoribosomes it is useful to start by determining the sedimentation profile of these structures by sucrose gradient centrifugation of mitochondrial extracts. During centrifugation, mitoribosome subunits, monosomes and polysomes, and potentially accumulated assembly intermediates will sediment through the gradient at different rates. Sedimentation will depend on the centrifugal force applied and the density and viscosity of the gradient. Importantly, it will also depend on the size, shape, and density of the mitoribosome particles present in the samples under study. Variations of this technique, often coupled with additional downstream approaches, have been used to analyze the process of mitoribosome biogenesis, the composition of assembly intermediates, or to monitor the interaction of extraribosomal proteins with individual mitoribosome subunits or monosomes.


Assuntos
Centrifugação com Gradiente de Concentração/métodos , RNA Helicases DEAD-box/química , Proteínas Mitocondriais/química , Ribossomos Mitocondriais/química , Proteínas Ribossômicas/química , Células HEK293 , Humanos , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/metabolismo , Fosforilação Oxidativa , Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo
16.
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
17.
Int J Mol Sci ; 22(1)2020 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-33374407

RESUMO

Thymosin α1 (Tα1) is an immunostimulatory peptide for the treatment of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections and used as an immune enhancer, which also offers prospects in the context of COVID-19 infections and cancer. Manufacturing of this N-terminally acetylated 28-residue peptide is demanding, and its short plasma half-life limits in vivo efficacy and requires frequent dosing. Here, we combined the PASylation technology with enzymatic in situ N-acetylation by RimJ to produce a long-acting version of Tα1 in Escherichia coli at high yield. ESI-MS analysis of the purified fusion protein indicated the expected composition without any signs of proteolysis. SEC analysis revealed a 10-fold expanded hydrodynamic volume resulting from the fusion with a conformationally disordered Pro/Ala/Ser (PAS) polypeptide of 600 residues. This size effect led to a plasma half-life in rats extended by more than a factor 8 compared to the original synthetic peptide due to retarded kidney filtration. Our study provides the basis for therapeutic development of a next generation thymosin α1 with prolonged circulation. Generally, the strategy of producing an N-terminally protected PASylated peptide solves three major problems of peptide drugs: (i) instability in the expression host, (ii) rapid degradation by serum exopeptidases, and (iii) low bioactivity because of fast renal clearance.


Assuntos
Adjuvantes Imunológicos/farmacocinética , Timalfasina/farmacocinética , Acetilação , Acetiltransferases/metabolismo , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/farmacologia , Animais , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Feminino , Meia-Vida , Espectrometria de Massas , Microscopia Eletrônica de Varredura , Neoplasias/tratamento farmacológico , Peptídeos/química , Proteólise , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/ultraestrutura , Proteínas Ribossômicas/metabolismo , Timalfasina/sangue , Timalfasina/química , Timalfasina/genética , Viroses/tratamento farmacológico
18.
PLoS One ; 15(10): e0235446, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33002000

RESUMO

We recently described a regulatory loop, which we termed autoregulation of infection (AOI), by which Sinorhizobium meliloti, a Medicago endosymbiont, downregulates the root susceptibility to secondary infection events via ethylene. AOI is initially triggered by so-far unidentified Medicago nodule signals named signal 1 and signal 1' whose transduction in bacteroids requires the S. meliloti outer-membrane-associated NsrA receptor protein and the cognate inner-membrane-associated adenylate cyclases, CyaK and CyaD1/D2, respectively. Here, we report on advances in signal 1 identification. Signal 1 activity is widespread as we robustly detected it in Medicago nodule extracts as well as in yeast and bacteria cell extracts. Biochemical analyses indicated a peptidic nature for signal 1 and, together with proteomic analyses, a universally conserved Medicago ribosomal protein of the uL2 family was identified as a candidate signal 1. Specifically, MtRPuL2A (MtrunA17Chr7g0247311) displays a strong signal activity that requires S. meliloti NsrA and CyaK, as endogenous signal 1. We have shown that MtRPuL2A is active in signaling only in a non-ribosomal form. A Medicago truncatula mutant in the major symbiotic transcriptional regulator MtNF-YA1 lacked most signal 1 activity, suggesting that signal 1 is under developmental control. Altogether, our results point to the MtRPuL2A ribosomal protein as the candidate for signal 1. Based on the Mtnf-ya1 mutant, we suggest a link between root infectiveness and nodule development. We discuss our findings in the context of ribosomal protein moonlighting.


Assuntos
Medicago truncatula , Proteínas de Plantas/metabolismo , Proteínas Ribossômicas/metabolismo , Nódulos Radiculares de Plantas/metabolismo , Sinorhizobium meliloti/metabolismo , Coinfecção/prevenção & controle , Etilenos/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Medicago truncatula/genética , Medicago truncatula/metabolismo , Medicago truncatula/microbiologia , Imunidade Vegetal/genética , Proteínas de Plantas/genética , Nodulação/fisiologia , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Proteínas Ribossômicas/genética , Nódulos Radiculares de Plantas/microbiologia , Transdução de Sinais , Simbiose
19.
Nat Commun ; 11(1): 5111, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037216

RESUMO

The nascent polypeptide exit tunnel (NPET) is a major functional center of 60S ribosomal subunits. However, little is known about how the NPET is constructed during ribosome assembly. We utilized molecular genetics, biochemistry, and cryo-electron microscopy (cryo-EM) to investigate the functions of two NPET-associated proteins, ribosomal protein uL4 and assembly factor Nog1, in NPET assembly. Structures of mutant pre-ribosomes lacking the tunnel domain of uL4 reveal a misassembled NPET, including an aberrantly flexible ribosomal RNA helix 74, resulting in at least three different blocks in 60S assembly. Structures of pre-ribosomes lacking the C-terminal extension of Nog1 demonstrate that this extension scaffolds the tunnel domain of uL4 in the NPET to help maintain stability in the core of pre-60S subunits. Our data reveal that uL4 and Nog1 work together in the maturation of ribosomal RNA helix 74, which is required to ensure proper construction of the NPET and 60S ribosomal subunits.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Microscopia Crioeletrônica , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Modelos Moleculares , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/genética , Domínios Proteicos , Estabilidade de RNA , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Eucariotos/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
20.
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
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