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1.
J Clin Invest ; 134(13)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38949021

RESUMO

Mechanical stress from cardiomyocyte contraction causes misfolded sarcomeric protein replacement. Sarcomeric maintenance utilizes localized pools of mRNAs and translation machinery, yet the importance of localized translation remains unclear. In this issue of the JCI, Haddad et al. identify the Z-line as a critical site for localized translation of sarcomeric proteins, mediated by ribosomal protein SA (RPSA). RPSA localized ribosomes at Z-lines and was trafficked via microtubules. Cardiomyocyte-specific loss of RPSA in mice resulted in mislocalized protein translation and caused structural dilation from myocyte atrophy. These findings demonstrate the necessity of RPSA-dependent spatially localized translation for sarcomere maintenance and cardiac structure and function.


Assuntos
Miócitos Cardíacos , Biossíntese de Proteínas , Proteínas Ribossômicas , Sarcômeros , Sarcômeros/metabolismo , Sarcômeros/patologia , Animais , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ribossomos/metabolismo , Ribossomos/genética , Humanos , Microtúbulos/metabolismo
2.
RNA Biol ; 21(1): 31-41, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38952121

RESUMO

Large ribosomal RNAs (rRNAs) are modified heavily post-transcriptionally in functionally important regions but, paradoxically, individual knockouts (KOs) of the modification enzymes have minimal impact on Escherichia coli growth. Furthermore, we recently constructed a strain with combined KOs of five modification enzymes (RluC, RlmKL, RlmN, RlmM and RluE) of the 'critical region' of the peptidyl transferase centre (PTC) in 23S rRNA that exhibited only a minor growth defect at 37°C (although major at 20°C). However, our combined KO of modification enzymes RluC and RlmE (not RluE) resulted in conditional lethality (at 20°C). Although the growth rates for both multiple-KO strains were characterized, the molecular explanations for such deficits remain unclear. Here, we pinpoint biochemical defects in these strains. In vitro fast kinetics at 20°C and 37°C with ribosomes purified from both strains revealed, counterintuitively, the slowing of translocation, not peptide bond formation or peptidyl release. Elongation rates of protein synthesis in vivo, as judged by the kinetics of ß-galactosidase induction, were also slowed. For the five-KO strain, the biggest deficit at 37°C was in 70S ribosome assembly, as judged by a dominant 50S peak in ribosome sucrose gradient profiles at 5 mM Mg2+. Reconstitution of this 50S subunit from purified five-KO rRNA and ribosomal proteins supported a direct role in ribosome biogenesis of the PTC region modifications per se, rather than of the modification enzymes. These results clarify the importance and roles of the enigmatic rRNA modifications.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Peptidil Transferases , Biossíntese de Proteínas , RNA Ribossômico , Ribossomos , Peptidil Transferases/metabolismo , Peptidil Transferases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Ribossomos/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Ribossômico 23S/metabolismo , RNA Ribossômico 23S/genética , Cinética
3.
PLoS One ; 19(6): e0304557, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38941348

RESUMO

Prenatal alcohol exposure (PAE) causes cognitive impairment and a distinctive craniofacial dysmorphology, due in part to apoptotic losses of the pluripotent cranial neural crest cells (CNCs) that form facial bones and cartilage. We previously reported that PAE rapidly represses expression of >70 ribosomal proteins (padj = 10-E47). Ribosome dysbiogenesis causes nucleolar stress and activates p53-MDM2-mediated apoptosis. Using primary avian CNCs and the murine CNC line O9-1, we tested whether nucleolar stress and p53-MDM2 signaling mediates this apoptosis. We further tested whether haploinsufficiency in genes that govern ribosome biogenesis, using a blocking morpholino approach, synergizes with alcohol to worsen craniofacial outcomes in a zebrafish model. In both avian and murine CNCs, pharmacologically relevant alcohol exposure (20mM, 2hr) causes the dissolution of nucleolar structures and the loss of rRNA synthesis; this nucleolar stress persisted for 18-24hr. This was followed by reduced proliferation, stabilization of nuclear p53, and apoptosis that was prevented by overexpression of MDM2 or dominant-negative p53. In zebrafish embryos, low-dose alcohol or morpholinos directed against ribosomal proteins Rpl5a, Rpl11, and Rps3a, the Tcof homolog Nolc1, or mdm2 separately caused modest craniofacial malformations, whereas these blocking morpholinos synergized with low-dose alcohol to reduce and even eliminate facial elements. Similar results were obtained using a small molecule inhibitor of RNA Polymerase 1, CX5461, whereas p53-blocking morpholinos normalized craniofacial outcomes under high-dose alcohol. Transcriptome analysis affirmed that alcohol suppressed the expression of >150 genes essential for ribosome biogenesis. We conclude that alcohol causes the apoptosis of CNCs, at least in part, by suppressing ribosome biogenesis and invoking a nucleolar stress that initiates their p53-MDM2 mediated apoptosis. We further note that the facial deficits that typify PAE and some ribosomopathies share features including reduced philtrum, upper lip, and epicanthal distance, suggesting the facial deficits of PAE represent, in part, a ribosomopathy.


Assuntos
Apoptose , Etanol , Crista Neural , Ribossomos , Proteína Supressora de Tumor p53 , Peixe-Zebra , Animais , Crista Neural/metabolismo , Crista Neural/efeitos dos fármacos , Ribossomos/metabolismo , Ribossomos/efeitos dos fármacos , Etanol/toxicidade , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Apoptose/efeitos dos fármacos , Camundongos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Nucléolo Celular/metabolismo , Nucléolo Celular/efeitos dos fármacos , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/genética , Crânio/patologia , Crânio/metabolismo , Crânio/efeitos dos fármacos , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
4.
Nat Commun ; 15(1): 5481, 2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-38942792

RESUMO

Tigecycline is widely used for treating complicated bacterial infections for which there are no effective drugs. It inhibits bacterial protein translation by blocking the ribosomal A-site. However, even though it is also cytotoxic for human cells, the molecular mechanism of its inhibition remains unclear. Here, we present cryo-EM structures of tigecycline-bound human mitochondrial 55S, 39S, cytoplasmic 80S and yeast cytoplasmic 80S ribosomes. We find that at clinically relevant concentrations, tigecycline effectively targets human 55S mitoribosomes, potentially, by hindering A-site tRNA accommodation and by blocking the peptidyl transfer center. In contrast, tigecycline does not bind to human 80S ribosomes under physiological concentrations. However, at high tigecycline concentrations, in addition to blocking the A-site, both human and yeast 80S ribosomes bind tigecycline at another conserved binding site restricting the movement of the L1 stalk. In conclusion, the observed distinct binding properties of tigecycline may guide new pathways for drug design and therapy.


Assuntos
Microscopia Crioeletrônica , Ribossomos , Tigeciclina , Tigeciclina/farmacologia , Tigeciclina/química , Humanos , Ribossomos/metabolismo , Ribossomos/efeitos dos fármacos , Antibacterianos/farmacologia , Antibacterianos/química , Sítios de Ligação , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos Mitocondriais/metabolismo , Ribossomos Mitocondriais/química , Ribossomos Mitocondriais/efeitos dos fármacos , Modelos Moleculares , RNA de Transferência/metabolismo , RNA de Transferência/química
5.
Mol Cell ; 84(12): 2337-2352.e9, 2024 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-38870935

RESUMO

Ribosome assembly requires precise coordination between the production and assembly of ribosomal components. Mutations in ribosomal proteins that inhibit the assembly process or ribosome function are often associated with ribosomopathies, some of which are linked to defects in proteostasis. In this study, we examine the interplay between several yeast proteostasis enzymes, including deubiquitylases (DUBs) Ubp2 and Ubp14, and E3 ligases Ufd4 and Hul5, and we explore their roles in the regulation of the cellular levels of K29-linked unanchored polyubiquitin (polyUb) chains. Accumulating K29-linked unanchored polyUb chains associate with maturing ribosomes to disrupt their assembly, activate the ribosome assembly stress response (RASTR), and lead to the sequestration of ribosomal proteins at the intranuclear quality control compartment (INQ). These findings reveal the physiological relevance of INQ and provide insights into mechanisms of cellular toxicity associated with ribosomopathies.


Assuntos
Poliubiquitina , Proteínas Ribossômicas , Ribossomos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/genética , Ribossomos/metabolismo , Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Poliubiquitina/metabolismo , Poliubiquitina/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Proteostase , Núcleo Celular/metabolismo
6.
Chem Rev ; 124(12): 7712-7730, 2024 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-38829723

RESUMO

The introduction of noncanonical amino acids into proteins has enabled researchers to modify fundamental physicochemical and functional properties of proteins. While the alteration of the genetic code, via the introduction of orthogonal aminoacyl-tRNA synthetase:tRNA pairs, has driven many of these efforts, the various components involved in the process of translation are important for the development of new genetic codes. In this review, we will focus on recent advances in engineering ribosomal machinery for noncanonical amino acid incorporation and genetic code modification. The engineering of the ribosome itself will be considered, as well as the many factors that interact closely with the ribosome, including both tRNAs and accessory factors, such as the all-important EF-Tu. Given the success of genome re-engineering efforts, future paths for radical alterations of the genetic code will require more expansive alterations in the translation machinery.


Assuntos
Aminoácidos , Código Genético , RNA de Transferência , Ribossomos , Aminoácidos/metabolismo , Aminoácidos/química , Ribossomos/metabolismo , RNA de Transferência/metabolismo , RNA de Transferência/genética , RNA de Transferência/química , Biossíntese de Proteínas , Engenharia de Proteínas , Aminoacil-tRNA Sintetases/metabolismo , Aminoacil-tRNA Sintetases/genética
7.
Microbiol Spectr ; 12(7): e0017024, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38832800

RESUMO

Diaphorin is a polyketide produced by "Candidatus Profftella armatura" (Gammaproteobacteria: Burkholderiales), an obligate symbiont of a devastating agricultural pest, the Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae). Physiological concentrations of diaphorin, which D. citri contains at levels as high as 2-20 mM, are inhibitory to various eukaryotes and Bacillus subtilis (Firmicutes: Bacilli) but promote the growth and metabolic activity of Escherichia coli (Gammaproteobacteria: Enterobacterales). Our previous study demonstrated that 5-mM diaphorin, which exhibits significant inhibitory and promoting effects on cultured B. subtilis and E. coli, respectively, inhibits in vitro gene expression utilizing purified B. subtilis and E. coli ribosomes. This suggested that the adverse effects of diaphorin on B. subtilis are partly due to its influence on gene expression. However, the result appeared inconsistent with the positive impact on E. coli. Moreover, the diaphorin concentration in bacterial cells, where genes are expressed in vivo, may be lower than in culture media. Therefore, the present study analyzed the effects of 50 and 500 µM of diaphorin on bacterial gene expression using the same analytical method. The result revealed that this concentration range of diaphorin, in contrast to 5-mM diaphorin, promotes the in vitro translation with the B. subtilis and E. coli ribosomes, suggesting that the positive effects of diaphorin on E. coli are due to its direct effects on translation. This study demonstrated for the first time that a pederin-type compound promotes gene expression, establishing a basis for utilizing its potential in pest management and industrial applications.IMPORTANCEThis study revealed that a limited concentration range of diaphorin, a secondary metabolite produced by a bacterial symbiont of an agricultural pest, promotes cell-free gene expression utilizing substrates and proteins purified from bacteria. The unique property of diaphorin, which is inhibitory to various eukaryotes and Bacillus subtilis but promotes the growth and metabolic activity of Escherichia coli, may affect the microbial flora of the pest insect, potentially influencing the transmission of devastating plant pathogens. Moreover, the activity may be exploited to improve the efficacy of industrial production by E. coli, which is often used to produce various important materials, including pharmaceuticals, enzymes, amino acids, and biofuels. This study elucidated a part of the mechanism by which the unique activity of diaphorin is expressed, constructing a foundation for applying the distinct property to pest management and industrial use.


Assuntos
Bacillus subtilis , Escherichia coli , Hemípteros , Policetídeos , Ribossomos , Simbiose , Hemípteros/microbiologia , Animais , Ribossomos/metabolismo , Ribossomos/genética , Policetídeos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Regulação Bacteriana da Expressão Gênica , Citrus/microbiologia , Gammaproteobacteria/genética , Gammaproteobacteria/metabolismo
8.
Genome Biol Evol ; 16(7)2024 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-38934859

RESUMO

During evolution, new open reading frames (ORFs) with the potential to give rise to novel proteins continuously emerge. A recent compilation of noncanonical ORFs with translation signatures in humans has identified thousands of cases with a putative de novo origin. However, it is not known which is their distribution in the population. Are they universally translated? Here, we use ribosome profiling data from 65 lymphoblastoid cell lines from individuals of Yoruba origin to investigate this question. We identify 2,587 de novo ORFs translated in at least one of the cell lines. In line with their de novo origin, the encoded proteins tend to be smaller than 100 amino acids and encode positively charged proteins. We observe that the de novo ORFs are more polymorphic in the population than the set of canonical proteins, with a substantial fraction of them being translated in only some of the cell lines. Remarkably, this difference remains significant after controlling for differences in the translation levels. These results suggest that variations in the level translation of de novo ORFs could be a relevant source of intraspecies phenotypic diversity in humans.


Assuntos
Fases de Leitura Aberta , Polimorfismo Genético , Humanos , Biossíntese de Proteínas , Linhagem Celular , Evolução Molecular , Ribossomos/genética , Ribossomos/metabolismo
9.
Life Sci Alliance ; 7(8)2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38858088

RESUMO

The signal recognition particle is essential for targeting transmembrane and secreted proteins to the endoplasmic reticulum. Remarkably, because they work together in the cytoplasm, the SRP and ribosomes are assembled in the same biomolecular condensate: the nucleolus. How important is the nucleolus for SRP assembly is not known. Using quantitative proteomics, we have investigated the interactomes of SRP components. We reveal that SRP proteins are associated with scores of nucleolar proteins important for ribosome biogenesis and nucleolar structure. Having monitored the subcellular distribution of SRP proteins upon controlled nucleolar disruption, we conclude that an intact organelle is required for their proper localization. Lastly, we have detected two SRP proteins in Cajal bodies, which indicates that previously undocumented steps of SRP assembly may occur in these bodies. This work highlights the importance of a structurally and functionally intact nucleolus for efficient SRP production and suggests that the biogenesis of SRP and ribosomes may be coordinated in the nucleolus by common assembly factors.


Assuntos
Nucléolo Celular , Proteômica , Ribossomos , Partícula de Reconhecimento de Sinal , Partícula de Reconhecimento de Sinal/metabolismo , Nucléolo Celular/metabolismo , Ribossomos/metabolismo , Humanos , Proteômica/métodos , Proteínas Nucleares/metabolismo , Corpos Enovelados/metabolismo , Células HeLa , Retículo Endoplasmático/metabolismo
10.
Elife ; 132024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38896445

RESUMO

The protein translocon at the endoplasmic reticulum comprises the Sec61 translocation channel and numerous accessory factors that collectively facilitate the biogenesis of secretory and membrane proteins. Here, we leveraged recent advances in cryo-electron microscopy (cryo-EM) and structure prediction to derive insights into several novel configurations of the ribosome-translocon complex. We show how a transmembrane domain (TMD) in a looped configuration passes through the Sec61 lateral gate during membrane insertion; how a nascent chain can bind and constrain the conformation of ribosomal protein uL22; and how the translocon-associated protein (TRAP) complex can adjust its position during different stages of protein biogenesis. Most unexpectedly, we find that a large proportion of translocon complexes contains RAMP4 intercalated into Sec61's lateral gate, widening Sec61's central pore and contributing to its hydrophilic interior. These structures lead to mechanistic hypotheses for translocon function and highlight a remarkably plastic machinery whose conformations and composition adjust dynamically to its diverse range of substrates.


Assuntos
Microscopia Crioeletrônica , Ribossomos , Canais de Translocação SEC , Ribossomos/metabolismo , Ribossomos/química , Ribossomos/ultraestrutura , Canais de Translocação SEC/metabolismo , Canais de Translocação SEC/química , Retículo Endoplasmático/metabolismo , Conformação Proteica , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/química , Humanos , Modelos Moleculares , Transporte Proteico , Proteínas de Membrana/metabolismo , Proteínas de Membrana/química
11.
Nat Commun ; 15(1): 4879, 2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38849354

RESUMO

The mammalian neocortex comprises an enormous diversity regarding cell types, morphology, and connectivity. In this work, we discover a post-transcriptional mechanism of gene expression regulation, protein translation, as a determinant of cortical neuron identity. We find specific upregulation of protein synthesis in the progenitors of later-born neurons and show that translation rates and concomitantly protein half-lives are inherent features of cortical neuron subtypes. In a small molecule screening, we identify Ire1α as a regulator of Satb2 expression and neuronal polarity. In the developing brain, Ire1α regulates global translation rates, coordinates ribosome traffic, and the expression of eIF4A1. Furthermore, we demonstrate that the Satb2 mRNA translation requires eIF4A1 helicase activity towards its 5'-untranslated region. Altogether, we show that cortical neuron diversity is generated by mechanisms operating beyond gene transcription, with Ire1α-safeguarded proteostasis serving as an essential regulator of brain development.


Assuntos
Proteínas de Ligação à Região de Interação com a Matriz , Neocórtex , Neurônios , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases , Animais , Neocórtex/metabolismo , Neocórtex/citologia , Neocórtex/embriologia , Neurônios/metabolismo , Neurônios/citologia , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteostase , Neurogênese/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Regiões 5' não Traduzidas/genética , Ribossomos/metabolismo , Ribossomos/genética , Humanos , Endorribonucleases/metabolismo , Endorribonucleases/genética , Diferenciação Celular/genética
12.
Sci Rep ; 14(1): 14253, 2024 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-38902339

RESUMO

The antibiotic fusidic acid (FA) is used to treat Staphylococcus aureus infections. It inhibits protein synthesis by binding to elongation factor G (EF-G) and preventing its release from the ribosome after translocation. While FA, due to permeability issues, is only effective against gram-positive bacteria, the available structures of FA-inhibited complexes are from gram-negative model organisms. To fill this knowledge gap, we solved cryo-EM structures of the S. aureus ribosome in complex with mRNA, tRNA, EF-G and FA to 2.5 Å resolution and the corresponding complex structures with the recently developed FA derivative FA-cyclopentane (FA-CP) to 2.0 Å resolution. With both FA variants, the majority of the ribosomal particles are observed in chimeric state and only a minor population in post-translocational state. As expected, FA binds in a pocket between domains I, II and III of EF-G and the sarcin-ricin loop of 23S rRNA. FA-CP binds in an identical position, but its cyclopentane moiety provides additional contacts to EF-G and 23S rRNA, suggesting that its improved resistance profile towards mutations in EF-G is due to higher-affinity binding. These high-resolution structures reveal new details about the S. aureus ribosome, including confirmation of many rRNA modifications, and provide an optimal starting point for future structure-based drug discovery on an important clinical drug target.


Assuntos
Microscopia Crioeletrônica , Ciclopentanos , Ácido Fusídico , Fator G para Elongação de Peptídeos , Ribossomos , Staphylococcus aureus , Ácido Fusídico/farmacologia , Ácido Fusídico/química , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , Ribossomos/metabolismo , Ribossomos/efeitos dos fármacos , Ciclopentanos/farmacologia , Ciclopentanos/química , Fator G para Elongação de Peptídeos/metabolismo , Fator G para Elongação de Peptídeos/química , Antibacterianos/farmacologia , Antibacterianos/química , Modelos Moleculares , RNA de Transferência/metabolismo , RNA de Transferência/química
14.
Mol Cell ; 84(11): 2135-2151.e7, 2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38848692

RESUMO

In response to stress, eukaryotes activate the integrated stress response (ISR) via phosphorylation of eIF2α to promote the translation of pro-survival effector genes, such as GCN4 in yeast. Complementing the ISR is the target of rapamycin (TOR) pathway, which regulates eIF4E function. Here, we probe translational control in the absence of eIF4E in Saccharomyces cerevisiae. Intriguingly, we find that loss of eIF4E leads to de-repression of GCN4 translation. In addition, we find that de-repression of GCN4 translation is accompanied by neither eIF2α phosphorylation nor reduction in initiator ternary complex (TC). Our data suggest that when eIF4E levels are depleted, GCN4 translation is de-repressed via a unique mechanism that may involve faster scanning by the small ribosome subunit due to increased local concentration of eIF4A. Overall, our findings suggest that relative levels of eIF4F components are key to ribosome dynamics and may play important roles in translational control of gene expression.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Estresse Fisiológico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fosforilação , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fator de Iniciação 4F em Eucariotos/metabolismo , Fator de Iniciação 4F em Eucariotos/genética , Biossíntese de Proteínas , Regulação Fúngica da Expressão Gênica , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Transdução de Sinais , Ribossomos/metabolismo , Ribossomos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Fator de Iniciação 4A em Eucariotos/genética
15.
Nat Commun ; 15(1): 4783, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38839776

RESUMO

Ribosomes translate the genetic code into proteins. Recent technical advances have facilitated in situ structural analyses of ribosome functional states inside eukaryotic cells and the minimal bacterium Mycoplasma. However, such analyses of Gram-negative bacteria are lacking, despite their ribosomes being major antimicrobial drug targets. Here we compare two E. coli strains, a lab E. coli K-12 and human gut isolate E. coli ED1a, for which tetracycline exhibits bacteriostatic and bactericidal action, respectively. Using our approach for close-to-native E. coli sample preparation, we assess the two strains by cryo-ET and visualize their ribosomes at high resolution in situ. Upon tetracycline treatment, these exhibit virtually identical drug binding sites, yet the conformation distribution of ribosomal complexes differs. While K-12 retains ribosomes in a translation-competent state, tRNAs are lost in the vast majority of ED1a ribosomes. These structural findings together with the proteome-wide abundance and thermal stability assessments indicate that antibiotic responses are complex in cells and can differ between different strains of a single species, thus arguing that all relevant bacterial strains should be analyzed in situ when addressing antibiotic mode of action.


Assuntos
Antibacterianos , Escherichia coli , Ribossomos , Tetraciclina , Ribossomos/metabolismo , Ribossomos/efeitos dos fármacos , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Tetraciclina/farmacologia , Microscopia Crioeletrônica , RNA de Transferência/metabolismo , RNA de Transferência/genética , Humanos , Sítios de Ligação , Biossíntese de Proteínas/efeitos dos fármacos , Escherichia coli K12/efeitos dos fármacos , Escherichia coli K12/genética , Escherichia coli K12/metabolismo
16.
Anal Biochem ; 692: 115580, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38825159

RESUMO

Ricin is one of the most toxic substances known and a type B biothreat agent. Shiga toxins (Stxs) produced by E. coli (STEC) and Shigella dysenteriae are foodborne pathogens. There is no effective therapy against ricin or STEC and there is an urgent need for inhibitors. Ricin toxin A subunit (RTA) and A1 subunit of Stx2a (Stx2A1) bind to the C-terminal domain (CTD) of the ribosomal P-stalk proteins to depurinate the sarcin/ricin loop. Modulation of toxin-ribosome interactions has not been explored as a strategy for inhibition. Therefore, development of assays that detect inhibitors targeting toxin-ribosome interactions remains a critical need. Here we describe a fluorescence anisotropy (FA)-based competitive binding assay using a BODIPY-TMR labeled 11-mer peptide (P11) derived from the P-stalk CTD to measure the binding affinity of peptides ranging from 3 to 11 amino acids for the P-stalk pocket of RTA and Stx2A1. Comparison of the affinity with the surface plasmon resonance (SPR) assay indicated that although the rank order was the same by both methods, the FA assay could differentiate better between peptides that show nonspecific interactions by SPR. The FA assay detects only interactions that compete with the labeled P11 and can validate inhibitor specificity and mechanism of action.


Assuntos
Polarização de Fluorescência , Ribossomos , Ricina , Ricina/antagonistas & inibidores , Ricina/metabolismo , Ricina/química , Polarização de Fluorescência/métodos , Ribossomos/metabolismo , Ressonância de Plasmônio de Superfície , Toxina Shiga/antagonistas & inibidores , Toxina Shiga/metabolismo , Toxina Shiga/química , Ligação Competitiva , Ligação Proteica , Toxina Shiga II/antagonistas & inibidores , Toxina Shiga II/metabolismo , Toxina Shiga II/química
17.
Life Sci Alliance ; 7(8)2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38866426

RESUMO

Multispanning membrane proteins are inserted into the endoplasmic reticulum membrane by the ribosome-bound multipass translocon (MPT) machinery. Based on cryo-electron tomography and extensive subtomogram analysis, we reveal the composition and arrangement of ribosome-bound MPT components in their native membrane environment. The intramembrane chaperone complex PAT and the translocon-associated protein (TRAP) complex associate substoichiometrically with the MPT in a translation-dependent manner. Although PAT is preferentially part of MPTs bound to translating ribosomes, the abundance of TRAP is highest in MPTs associated with non-translating ribosomes. The subtomogram average of the TRAP-containing MPT reveals intermolecular contacts between the luminal domains of TRAP and an unknown subunit of the back-of-Sec61 complex. AlphaFold modeling suggests this protein is nodal modulator, bridging the luminal domains of nicalin and TRAPα. Collectively, our results visualize the variability of MPT factors in the native membrane environment dependent on the translational activity of the bound ribosome.


Assuntos
Retículo Endoplasmático , Proteínas de Membrana , Ribossomos , Ribossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/química , Retículo Endoplasmático/metabolismo , Biossíntese de Proteínas , Microscopia Crioeletrônica , Canais de Translocação SEC/metabolismo , Canais de Translocação SEC/química , Chaperonas Moleculares/metabolismo , Transporte Proteico , Modelos Moleculares
18.
Brief Bioinform ; 25(4)2024 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-38842510

RESUMO

Accurate and comprehensive annotation of microprotein-coding small open reading frames (smORFs) is critical to our understanding of normal physiology and disease. Empirical identification of translated smORFs is carried out primarily using ribosome profiling (Ribo-seq). While effective, published Ribo-seq datasets can vary drastically in quality and different analysis tools are frequently employed. Here, we examine the impact of these factors on identifying translated smORFs. We compared five commonly used software tools that assess open reading frame translation from Ribo-seq (RibORFv0.1, RibORFv1.0, RiboCode, ORFquant, and Ribo-TISH) and found surprisingly low agreement across all tools. Only ~2% of smORFs were called translated by all five tools, and ~15% by three or more tools when assessing the same high-resolution Ribo-seq dataset. For larger annotated genes, the same analysis showed ~74% agreement across all five tools. We also found that some tools are strongly biased against low-resolution Ribo-seq data, while others are more tolerant. Analyzing Ribo-seq coverage revealed that smORFs detected by more than one tool tend to have higher translation levels and higher fractions of in-frame reads, consistent with what was observed for annotated genes. Together these results support employing multiple tools to identify the most confident microprotein-coding smORFs and choosing the tools based on the quality of the dataset and the planned downstream characterization experiments of the predicted smORFs.


Assuntos
Fases de Leitura Aberta , Software , Ribossomos/metabolismo , Ribossomos/genética , Anotação de Sequência Molecular/métodos , Humanos , Biossíntese de Proteínas , Biologia Computacional/métodos , Perfil de Ribossomos
19.
Nucleus ; 15(1): 2373052, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38940456

RESUMO

The analysis of nucleocytoplasmic transport of proteins and messenger RNA has been the focus of advanced microscopic approaches. Recently, it has been possible to identify and visualize individual pre-ribosomal particles on their way through the nuclear pore complex using both electron and light microscopy. In this review, we focused on the transport of pre-ribosomal particles in the nucleus on their way to and through the pores.


Assuntos
Transporte Ativo do Núcleo Celular , Nucléolo Celular , Citoplasma , Poro Nuclear , Nucléolo Celular/metabolismo , Poro Nuclear/metabolismo , Citoplasma/metabolismo , Humanos , Animais , Ribossomos/metabolismo , Núcleo Celular/metabolismo
20.
Int J Mol Sci ; 25(11)2024 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-38892101

RESUMO

The central dogma treats the ribosome as a molecular machine that reads one mRNA codon at a time as it adds each amino acid to its growing peptide chain. However, this and previous studies suggest that ribosomes actually perceive pairs of adjacent codons as they take three-nucleotide steps along the mRNA. We examined GNN codons, which we find are surprisingly overrepresented in eukaryote protein-coding open reading frames (ORFs), especially immediately after NNU codons. Ribosome profiling experiments in yeast revealed that ribosomes with NNU at their aminoacyl (A) site have particularly elevated densities when NNU is immediately followed (3') by a GNN codon, indicating slower mRNA threading of the NNU codon from the ribosome's A to peptidyl (P) sites. Moreover, if the assessment was limited to ribosomes that have only recently arrived at the next codon, by examining 21-nucleotide ribosome footprints (21-nt RFPs), elevated densities were observed for multiple codon classes when followed by GNN. This striking translation slowdown at adjacent 5'-NNN GNN codon pairs is likely mediated, in part, by the ribosome's CAR surface, which acts as an extension of the A-site tRNA anticodon during ribosome translocation and interacts through hydrogen bonding and pi stacking with the GNN codon. The functional consequences of 5'-NNN GNN codon adjacency are expected to influence the evolution of protein coding sequences.


Assuntos
Códon , Fases de Leitura Aberta , Biossíntese de Proteínas , RNA Mensageiro , Ribossomos , Códon/genética , Ribossomos/metabolismo , Ribossomos/genética , Fases de Leitura Aberta/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Anticódon/genética
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