RESUMO
Epithelial-to-mesenchymal transition (EMT) contributes significantly to chemotherapy resistance and remains a critical challenge in treating advanced breast cancer. The complexity of EMT, involving redundant pro-EMT signaling pathways and its paradox reversal process, mesenchymal-to-epithelial transition (MET), has hindered the development of effective treatments. In this study, we utilized a Tri-PyMT EMT lineage-tracing model in mice and single-cell RNA sequencing (scRNA-seq) to comprehensively analyze the EMT status of tumor cells. Our findings revealed elevated ribosome biogenesis (RiBi) during the transitioning phases of both EMT and MET processes. RiBi and its subsequent nascent protein synthesis mediated by ERK and mTOR signalings are essential for EMT/MET completion. Importantly, inhibiting excessive RiBi genetically or pharmacologically impaired the EMT/MET capability of tumor cells. Combining RiBi inhibition with chemotherapy drugs synergistically reduced metastatic outgrowth of epithelial and mesenchymal tumor cells under chemotherapies. Our study suggests that targeting the RiBi pathway presents a promising strategy for treating patients with advanced breast cancer.
Although there have been considerable improvements in breast cancer treatments over the years, there are still many patients whose cancerous cells become resistant to treatments, including chemotherapy. Several different factors can contribute to resistance to chemotherapy, but one important change is the epithelial-to-mesenchymal transition (or EMT for short). During this transition, breast cancer cells become more aggressive, and more able to metastasize and spread to other parts of the body. Cells can also go through the reverse process called the mesenchymal-to-epithelial transition (or MET for short). Together, EMT and MET help breast cancer cells become resilient to treatment. However, it was not clear if these transitions shared a mechanism or pathway that could be targeted as a way to make cancer treatments more effective. To investigate, Ban, Zou et al. studied breast cancer cells from mice which had been labelled with fluorescent proteins that indicated whether a cell had ever transitioned between an epithelial and mesenchymal state. Various genetic experiments revealed that breast cancer cells in the EMT or MET phase made a lot more ribosomes, molecules that are vital for producing new proteins. Ban, Zhou et al. found that blocking the production of ribosomes (using drugs or genetic tools) prevented the cells from undergoing both EMT and MET. Further experiments showed that when mice with breast cancer were treated with a standard chemotherapy treatment plus an anti-ribosome drug, this reduced the number and size of tumors that had metastasized to the lung. This suggests that blocking ribosome production makes breast cancer cells undergoing EMT and/or MET less resistant to chemotherapy. Future studies will have to ascertain whether these findings also apply to patients with breast cancer. In particular, one of the drugs used to block ribosome production in this study is in early-phase clinical trials, so future trials may be able to assess the drug's effect in combination with chemotherapies.
Assuntos
Neoplasias da Mama , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Ribossomos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Camundongos , Feminino , Ribossomos/metabolismo , Ribossomos/efeitos dos fármacos , Humanos , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biogênese de Organelas , Transdução de Sinais/efeitos dos fármacosRESUMO
SETD8 is a methyltransferase that is overexpressed in several cancers, which monomethylates H4K20 as well as other non-histone targets such as PCNA or p53. We here report novel SETD8 inhibitors, which were discovered while trying to identify chemicals that prevent 53BP1 foci formation, an event mediated by H4K20 methylation. Consistent with previous reports, SETD8 inhibitors induce p53 expression, although they are equally toxic for p53 proficient or deficient cells. Thermal stability proteomics revealed that the compounds had a particular impact on nucleoli, which was confirmed by fluorescent and electron microscopy. Similarly, Setd8 deletion generated nucleolar stress and impaired ribosome biogenesis, supporting that this was an on-target effect of SETD8 inhibitors. Furthermore, a genome-wide CRISPR screen identified an enrichment of nucleolar factors among those modulating the toxicity of SETD8 inhibitors. Accordingly, the toxicity of SETD8 inhibition correlated with MYC or mTOR activity, key regulators of ribosome biogenesis. Together, our study provides a new class of SETD8 inhibitors and a novel biomarker to identify tumors most likely to respond to this therapy.
Assuntos
Histona-Lisina N-Metiltransferase , Ribossomos , Humanos , Ribossomos/metabolismo , Ribossomos/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Linhagem Celular Tumoral , Nucléolo Celular/metabolismo , Nucléolo Celular/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neoplasias/metabolismo , Neoplasias/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Serina-Treonina Quinases TOR/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
The emergence of the "super fungus" Candida auris poses a significant threat to human health, given its multidrug resistance and high mortality rates. Therefore, developing a new antifungal strategy is necessary. Our previous research showed that Baicalein (BE), a key bioactive compound from the dried root of the perennial herb Scutellaria baicalensis Georgi, has strong fungistatic properties against C. auris. Nevertheless, the antifungal activity of BE against C. auris and its mechanism of action requires further investigation. In this study, we explored how BE affects this fungus using various techniques, including scanning electron microscopy (SEM), Annexin V-FITC apoptosis detection, CaspACE FITC-VAD-FMK In Situ Marker, reactive oxygen species (ROS) assay, singlet oxygen sensor green (SOSG) fluorescent probe, enhanced mitochondrial membrane potential (MMP) assay with JC-1, DAPI staining, TUNEL assay and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Our findings revealed that BE induced several apoptotic features, including phosphatidylserine (PS) externalization, metacaspase activation, nuclear condensation and DNA fragmentation. BE also increased intracellular ROS levels and altered mitochondrial functions. Additionally, transcriptomic analysis and RT-qPCR validation indicated that BE may induce apoptosis in C. auris by affecting ribosome-related pathways, suggesting that ribosomes could be new targets for antifungal agents, in addition to cell walls, membranes, and DNA. This study emphasizes the antifungal activity and mechanism of BE against C. auris, offering a promising treatment strategy for C. auris infection.
Assuntos
Antifúngicos , Apoptose , Candida , Flavanonas , Potencial da Membrana Mitocondrial , Espécies Reativas de Oxigênio , Ribossomos , Flavanonas/farmacologia , Apoptose/efeitos dos fármacos , Candida/efeitos dos fármacos , Antifúngicos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Testes de Sensibilidade Microbiana , HumanosRESUMO
BACKGROUND: Macrolides inhibit the growth of bacterial cells by preventing the elongation of polypeptides during protein biosynthesis and include natural, synthetic, and semi-synthetic products. Elongation prevention occurs by blocking the passage of the polypeptide chain as the macrolides bind at the nascent peptide exit tunnel. OBJECTIVE: Recent data of ribosome profiling via ribo-seq further proves that, other than blocking the polypeptide chain, macrolides are also able to affect the synthesis of individual proteins. Thus, this shows that the mode of action of macrolides is more complex than we initially thought. Since the discovery of macrolides in the 1950s, they have been widely used in veterinary practice, agriculture, and medicine. Due to misuse and overuse of antibiotics, bacteria have acquired resistance against them. Hence, it is of utmost importance for us to fully understand the mode of action of macrolides as well as the mechanisms of resistance against macrolides in order to mitigate antibiotic-resistance issues. RESULTS: Chemical modifications can be performed to improve macrolide potency if we have a better understanding of their mode of action. Furthermore, a complete and detailed understanding of the mode of action of macrolides has remained vague, as new findings have challenged theories that are already in existence-due to this obscurity, research into macrolide modes of action continues to this day. CONCLUSION: In this review, we present an overview of macrolide antibiotics, with an emphasis on the latest knowledge regarding the mode of action of macrolides as well as the mechanisms of resistance employed by bacteria against macrolides.
Assuntos
Antibacterianos , Bactérias , Farmacorresistência Bacteriana , Macrolídeos , Macrolídeos/farmacologia , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/genética , Humanos , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacosRESUMO
Elastin, a key structural protein essential for the elasticity of the skin and elastogenic tissues, degrades with age. Replenishing elastin holds promise for anti-aging cosmetics and the supplementation of elastic activities of the cardiovascular system. We employed RiboScreenTM, a technology for identifying molecules that enhance the production of specific proteins, to target the production of tropoelastin. We make use of RiboScreenTM in two crucial steps: first, to pinpoint a target ribosomal protein (TRP), which acts as a switch to increase the production of the protein of interest (POI), and second, to identify small molecules that activate this ribosomal protein switch. Using RiboScreenTM, we identified ribosomal protein L40, henceforth eL40, as a TRP switch to boost tropoelastin production. Drug discovery identified a small-molecule hit that binds to eL40. In-cell treatment demonstrated activity of the eL40 ligand and delivered increased tropoelastin production levels in a dose-dependent manner. Thus, we demonstrate that RiboScreenTM can successfully identify a small-molecule hit capable of selectively enhancing tropoelastin production. This compound has the potential to be developed for topical or systemic applications to promote skin rejuvenation and to supplement elastic functionality within the cardiovascular system.
Assuntos
Elastina , Proteínas Ribossômicas , Ribossomos , Tropoelastina , Tropoelastina/metabolismo , Tropoelastina/genética , Humanos , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/genética , Elastina/metabolismo , Elastina/genética , Ribossomos/metabolismo , Ribossomos/efeitos dos fármacos , Ligantes , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Bacteria often evolve antibiotic resistance through mutagenesis. However, the processes causing the mutagenesis have not been fully resolved. Here, we find that a broad range of ribosome-targeting antibiotics cause mutations through an underexplored pathway. Focusing on the clinically important aminoglycoside gentamicin, we find that the translation inhibitor causes genome-wide premature stalling of RNA polymerase (RNAP) in a loci-dependent manner. Further analysis shows that the stalling is caused by the disruption of transcription-translation coupling. Anti-intuitively, the stalled RNAPs subsequently induce lesions to the DNA via transcription-coupled repair. While most of the bacteria are killed by genotoxicity, a small subpopulation acquires mutations via SOS-induced mutagenesis. Given that these processes are triggered shortly after antibiotic addition, resistance rapidly emerges in the population. Our work reveals a mechanism of action of ribosomal antibiotics, illustrates the importance of dissecting the complex interplay between multiple molecular processes in understanding antibiotic efficacy, and suggests new strategies for countering the development of resistance.
Assuntos
Antibacterianos , RNA Polimerases Dirigidas por DNA , Farmacorresistência Bacteriana , Instabilidade Genômica , Gentamicinas , Ribossomos , Antibacterianos/farmacologia , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Ribossomos/metabolismo , Ribossomos/efeitos dos fármacos , Gentamicinas/farmacologia , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Mutação , Mutagênese , Transcrição Gênica/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacosRESUMO
The formation of new ribosomes is tightly coordinated with cell growth and proliferation. In eukaryotes, the correct assembly of all ribosomal proteins and RNAs follows an intricate scheme of maturation and rearrangement steps across three cellular compartments: the nucleolus, nucleoplasm, and cytoplasm. We demonstrate that usnic acid, a lichen secondary metabolite, inhibits the maturation of the large ribosomal subunit in yeast. We combine biochemical characterization of pre-ribosomal particles with a quantitative single-particle cryo-EM approach to monitor changes in nucleolar particle populations upon drug treatment. Usnic acid rapidly blocks the transition from nucleolar state B to C of Nsa1-associated pre-ribosomes, depleting key maturation factors such as Dbp10 and hindering pre-rRNA processing. This primary nucleolar block rapidly rebounds on earlier stages of the pathway which highlights the regulatory linkages between different steps. In summary, we provide an in-depth characterization of the effect of usnic acid on ribosome biogenesis, which may have implications for its reported anti-cancer activities.
Assuntos
Benzofuranos , Nucléolo Celular , Microscopia Crioeletrônica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Benzofuranos/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Nucléolo Celular/metabolismo , Nucléolo Celular/efeitos dos fármacos , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Ribossomos/efeitos dos fármacos , RNA Ribossômico/metabolismo , Subunidades Ribossômicas Maiores/metabolismo , Precursores de RNA/metabolismo , Precursores de RNA/genética , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Líquens/metabolismoRESUMO
Chloramphenicol (CHL) is an antibiotic targeting the peptidyl transferase center in bacterial ribosomes. We synthesized a new analog, CAM-BER, by substituting the dichloroacetyl moiety of CHL with a positively charged aromatic berberine group. CAM-BER suppresses bacterial cell growth, inhibits protein synthesis in vitro, and binds tightly to the 70S ribosome. Crystal structure analysis reveals that the bulky berberine group folds into the P site of the peptidyl transferase center (PTC), where it competes with the formyl-methionine residue of the initiator tRNA. Our toe-printing data confirm that CAM-BER acts as a translation initiation inhibitor in stark contrast to CHL, a translation elongation inhibitor. Moreover, CAM-BER induces a distinct rearrangement of conformationally restrained nucleotide A2059, suggesting that the 23S rRNA plasticity is significantly higher than previously thought. CAM-BER shows potential in avoiding CHL resistance and presents opportunities for developing novel berberine derivatives of CHL through medicinal chemistry exploration.
Assuntos
Berberina , Cloranfenicol , Ribossomos , Cloranfenicol/farmacologia , Cloranfenicol/química , Cloranfenicol/metabolismo , Berberina/farmacologia , Berberina/química , Berberina/análogos & derivados , Berberina/metabolismo , Ribossomos/metabolismo , Ribossomos/efeitos dos fármacos , Cristalografia por Raios X , Antibacterianos/farmacologia , Antibacterianos/química , Modelos Moleculares , Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Sítios de Ligação , RNA Ribossômico 23S/metabolismo , RNA Ribossômico 23S/química , Peptidil Transferases/metabolismo , Peptidil Transferases/química , Biossíntese de Proteínas/efeitos dos fármacos , Conformação de Ácido NucleicoRESUMO
Shiga toxins are the main virulence factors of Shiga toxin producing E. coli (STEC) and S. dysenteriae. There is no effective therapy to counter the disease caused by these toxins. The A1 subunits of Shiga toxins bind the C-termini of ribosomal P-stalk proteins to depurinate the sarcin/ricin loop. The ribosome binding site of Shiga toxin 2 has not been targeted by small molecules. We screened a fragment library against the A1 subunit of Shiga toxin 2 (Stx2A1) and identified a fragment, BTB13086, which bound at the ribosome binding site and mimicked the binding mode of the P-stalk proteins. We synthesized analogs of BTB13086 and identified a series of molecules with similar affinity and inhibitory activity. These are the first compounds that bind at the ribosome binding site of Stx2A1 and inhibit activity. These compounds hold great promise for further inhibitor development against STEC infection.
Assuntos
Ribossomos , Toxina Shiga II , Ribossomos/metabolismo , Ribossomos/efeitos dos fármacos , Toxina Shiga II/antagonistas & inibidores , Toxina Shiga II/metabolismo , Toxina Shiga II/química , Sítios de Ligação , Ligação Proteica , Escherichia coli Shiga Toxigênica/efeitos dos fármacos , Escherichia coli Shiga Toxigênica/metabolismo , HumanosRESUMO
Spectinomycin is an aminocyclitol antibiotic with a unique ribosomal binding site. Prior synthetic modifications of spectinomycin have enhanced potency and antibacterial spectrum through addition at the 6'-position to produce trospectomycin and to the 3'-position to produce spectinamides and aminomethyl spectinomycins. This study focused on the design, synthesis, and evaluation of three 3',6'-disubstituted spectinomycin analogs: trospectinamide, N-benzyl linked aminomethyl, and N-ethylene linked aminomethyl trospectomycins. Computational experiments predicted that these disubstituted analogs would be capable of binding within the SPC ribosomal binding site. The new analogs were synthesized from trospectomycin, adapting the previously established routes for the spectinamide and aminomethyl spectinomycin series. In a cell-free translation assay, the disubstituted analogs showed ribosomal inhibition similar to spectinomycin or trospectomycin. These disubstituted analogs demonstrated inhibitory MIC activity against various bacterial species with the 3'-modification dictating spectrum of activity, leading to improved activity against mycobacterium species. Notably, N-ethylene linked aminomethyl trospectomycins exhibited increased potency against Mycobacterium abscessus and trospectinamide displayed robust activity against M. tuberculosis, aligning with the selective efficacy of spectinamides. The study also found that trospectomycin is susceptible to efflux in M. tuberculosis and M. abscessus. These findings contribute to the understanding of the structure-activity relationship of spectinomycin analogs and can guide the design and synthesis of more effective spectinomycin compounds.
Assuntos
Antibacterianos , Testes de Sensibilidade Microbiana , Espectinomicina , Espectinomicina/farmacologia , Espectinomicina/análogos & derivados , Espectinomicina/síntese química , Antibacterianos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Relação Estrutura-Atividade , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Sítios de Ligação , Bactérias/efeitos dos fármacosRESUMO
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/químicaRESUMO
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/metabolismoRESUMO
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/metabolismoRESUMO
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/químicaRESUMO
OBJECTIVES: Mycobacterium abscessus is a non-tuberculous mycobacterial pathogen that causes pulmonary and skin infections globally. Clarithromycin plays a pivotal role in treating M. abscessus infections, with resistance often leading to treatment failure. While canonical mutations in the 23S rRNA residue 2270/2271 are recognized as the primary mechanism for acquired clarithromycin resistance, resistant isolates lacking these mutations have been widely reported. This study aims to identify new mechanisms of clarithromycin resistance in M. abscessus. METHODS: We selected spontaneous resistant mutants derived from two parental strains characterized by erm(41) T28 and C28 sequevars, respectively. Whole-genome sequencing was performed on mutants lacking the 23S rRNA 2270/2271 mutations. Site-directed mutagenesis was used to confirm the resistance phenotypes of newly identified mutations. Bioinformatic analysis of publicly available genomes was conducted to evaluate the presence of these mutations in clinical isolates. The spatial localization of these mutations in the ribosome was analyzed to investigate potential mechanisms of resistance. RESULTS: A total of 135 resistant mutants were selected from the parental strains. Sequencing of the 78 mutants lacking the 23S rRNA 2270/2271 mutations identified mutations within the peptidyl-transferase center and hairpin loops 35, 49, and 74 of the 23S rRNA. These noncanonical mutations were identified in 57 of 1875 genomes of clinical isolates. Thirteen representative mutations were introduced into the bacterial genome, and their contributions to macrolide resistance were confirmed. The newly identified mutations all localized at the entrance of the nascent peptide exit tunnel, potentially contributing to resistance by disrupting the macrolide binding pocket. CONCLUSION: Several noncanonical 23S rRNA mutations conferring clarithromycin resistance were identified. These mutations enhance our understanding of macrolide resistance in M. abscessus and could serve as important markers for diagnosing clarithromycin resistance.
Assuntos
Antibacterianos , Claritromicina , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium abscessus , RNA Ribossômico 23S , Ribossomos , Claritromicina/farmacologia , Mycobacterium abscessus/genética , Mycobacterium abscessus/efeitos dos fármacos , RNA Ribossômico 23S/genética , Farmacorresistência Bacteriana/genética , Antibacterianos/farmacologia , Ribossomos/efeitos dos fármacos , Ribossomos/genética , Ribossomos/metabolismo , Humanos , Infecções por Mycobacterium não Tuberculosas/microbiologia , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Sequenciamento Completo do Genoma , Mutagênese Sítio-DirigidaRESUMO
A growing number of studies have demonstrated that repeated exposure to sevoflurane during development results in persistent social abnormalities and cognitive impairment. Davunetide, an active fragment of the activity-dependent neuroprotective protein (ADNP), has been implicated in social and cognitive protection. However, the potential of davunetide to attenuate social deficits following sevoflurane exposure and the underlying developmental mechanisms remain poorly understood. In this study, ribosome and proteome profiles were analyzed to investigate the molecular basis of sevoflurane-induced social deficits in neonatal mice. The neuropathological basis was also explored using Golgi staining, morphological analysis, western blotting, electrophysiological analysis, and behavioral analysis. Results indicated that ADNP was significantly down-regulated following developmental exposure to sevoflurane. In adulthood, anterior cingulate cortex (ACC) neurons exposed to sevoflurane exhibited a decrease in dendrite number, total dendrite length, and spine density. Furthermore, the expression levels of Homer, PSD95, synaptophysin, and vglut2 were significantly reduced in the sevoflurane group. Patch-clamp recordings indicated reductions in both the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs). Notably, davunetide significantly ameliorated the synaptic defects, social behavior deficits, and cognitive impairments induced by sevoflurane. Mechanistic analysis revealed that loss of ADNP led to dysregulation of Ca 2+ activity via the Wnt/ß-catenin signaling, resulting in decreased expression of synaptic proteins. Suppression of Wnt signaling was restored in the davunetide-treated group. Thus, ADNP was identified as a promising therapeutic target for the prevention and treatment of neurodevelopmental toxicity caused by general anesthetics. This study provides important insights into the mechanisms underlying social and cognitive disturbances caused by sevoflurane exposure in neonatal mice and elucidates the regulatory pathways involved.
Assuntos
Disfunção Cognitiva , Proteínas do Tecido Nervoso , Proteoma , Ribossomos , Sevoflurano , Comportamento Social , Animais , Masculino , Camundongos , Anestésicos Inalatórios/efeitos adversos , Anestésicos Inalatórios/toxicidade , Anestésicos Inalatórios/farmacologia , Animais Recém-Nascidos , Comportamento Animal/efeitos dos fármacos , Disfunção Cognitiva/induzido quimicamente , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismoRESUMO
The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the 'WIN' site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small-molecule WINi, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anticancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in human MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anticancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Proteína de Leucina Linfoide-Mieloide , Proteínas Nucleares , Ribossomos , Proteína Supressora de Tumor p53 , Humanos , Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Peptidomiméticos/farmacologiaRESUMO
Ribosome-inactivating proteins (RIPs) are a group of proteins with rRNA N-glycosylase activity that irreversibly inhibit protein synthesis and consequently cause cell death. Recently, an RIP called ledodin has been found in shiitake; it is cytotoxic, strongly inhibits protein synthesis, and shows rRNA N-glycosylase activity. In this work, we isolated and characterized a 50 kDa cytotoxic protein from shiitake that we named edodin. Edodin inhibits protein synthesis in a mammalian cell-free system, but not in insect-, yeast-, and bacteria-derived systems. It exhibits rRNA N-glycosylase and DNA-nicking activities, which relate it to plant RIPs. It was also shown to be toxic to HeLa and COLO 320 cells. Its structure is not related to other RIPs found in plants, bacteria, or fungi, but, instead, it presents the characteristic structure of the fold type I of pyridoxal phosphate-dependent enzymes. Homologous sequences have been found in other fungi of the class Agaricomycetes; thus, edodin could be a new type of toxin present in many fungi, some of them edible, which makes them of great interest in health, both for their involvement in food safety and for their potential biomedical and biotechnological applications.
Assuntos
Ribossomos , Cogumelos Shiitake , Humanos , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Cogumelos Shiitake/química , Células HeLa , Animais , Micotoxinas/toxicidade , Micotoxinas/química , Proteínas Inativadoras de Ribossomos/química , Proteínas Inativadoras de Ribossomos/farmacologia , Proteínas Fúngicas/química , Proteínas Fúngicas/toxicidade , Proteínas Fúngicas/farmacologia , Proteínas Fúngicas/metabolismo , Linhagem Celular TumoralRESUMO
Macrozones are novel conjugates of azithromycin and thiosemicarbazones, which exhibit very good in vitro antibacterial activities against susceptible and some resistant bacterial strains thus showing a potential for further development. A combination of spectrometric (fluorimetry, STD and WaterLOGSY NMR) and molecular docking studies provided insights into atomic details of interactions between selected macrozones and biological receptors such as E. coli ribosome and bovine serum albumin. Fluorimetric measurements revealed binding constants in the micro-molar range while NMR experiments provided data on binding epitopes. It has been demonstrated that both STD and WaterLOGSY gave comparable and consistent results unveiling atoms in intimate contacts with biological receptors. Docking studies pointed towards main interactions between macrozones and E. coli ribosome which included specific π - π stacking and hydrogen bonding interactions with thiosemicarbazone part extending down the ribosome exit tunnel. The results of the docking experiments were in fine correlation with those obtained by NMR and fluorimetry. Our investigation pointed towards a two-site binding mechanism of interactions between macrozones and E. coli ribosome which is the most probable reason for their activity against azithromycin-resistant strains. Much better activity of macrozone-nickel coordinated compound against E. coli ribosome compared to other macrozones has been attributed to the higher polarity which enabled better bacterial membrane penetration and binding of the two thiosemicarbazone units thus additionally contributing to the overall binding energy. The knowledge gained in this study should play an important role in anti-infective macrolide design in the future.
Assuntos
Antibacterianos , Escherichia coli , Fluorometria , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Antibacterianos/farmacologia , Antibacterianos/química , Escherichia coli/efeitos dos fármacos , Sítios de Ligação , Estrutura Molecular , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Tiossemicarbazonas/química , Tiossemicarbazonas/farmacologia , Relação Estrutura-Atividade , Ribossomos/metabolismo , Ribossomos/efeitos dos fármacos , Relação Dose-Resposta a Droga , Animais , Bovinos , Azitromicina/farmacologia , Azitromicina/química , Azitromicina/metabolismoRESUMO
The bacterial ribosome is an essential drug target as many clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent resistance mechanisms to PTC-acting drugs in Gram-positive bacteria is C8-methylation of the universally conserved A2503 nucleobase by Cfr methylase in 23S ribosomal RNA. Despite its clinical importance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. Here, we report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-transfer RNAs. These structures reveal an allosteric rearrangement of nucleotide A2062 upon Cfr-mediated methylation of A2503 that likely contributes to the reduced potency of some PTC inhibitors. Additionally, we provide the structural bases behind two distinct mechanisms of engaging the Cfr-methylated ribosome by the antibiotics iboxamycin and tylosin.