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Inflammation and the Gram-positive anaerobic bacterium Cutibacterium acnes, which is implicated in acne pathogenesis and pilosebaceous-unit inflammation, are the main targets of antibiotic-based therapy against acne vulgaris (acne). The most widely used antibiotics in acne therapy are tetracyclines, macrolides, and lincosamides. Unfortunately, C. acnes bacteria over the past several decades have demonstrated increased resistance to these antibiotics, particularly to clindamycin. The precise knowledge of how antibiotics interact with their clinical target is needed to overcome this problem. Toward this goal, we determined the structure of clindamycin in complex with the ribosome of C. acnes at 2.53 Å resolution using cryogenic electron microscopy. The galactose sugar moiety of clindamycin interacts with nucleotides of the 23S ribosomal RNA directly or through a conserved network of water-mediated interactions. Its propyl pyrrolidinyl group interacts with the 23S ribosomal RNA through van der Waals forces. Clindamycin binding to the C. acnes ribosome interferes with both: proper orientation of the aminoacyl group of the A-site bound transfer RNA that is needed for peptide bond formation and with the extension of the nascent peptide. Our data are important for advancing the understanding of antibiotic resistance and development of narrow-spectrum antibacterial drugs, which is an urgent need for contemporary antibiotic stewardship., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Bile acids (BAs) exert a profound influence on the body's pathophysiology by intricately shaping the composition of gut bacteria. However, the complex interplay between BAs and gut microbiota has impeded a systematic exploration of their impact on intestinal bacteria. Initially, we investigated the effects of 21 BAs on the growth of 65 gut bacterial strains in vitro. Subsequently, we examined the impact of BAs on the overall composition of intestinal bacteria, both in vivo and in vitro. The results unveiled distinct effects of various BAs on different intestinal strains and their diverse impacts on the composition of gut bacteria. Mechanistically, the inhibition of intestinal strains by BAs occurs through the accumulation of these acids within the strains. The intracellular accumulation of deoxycholic acid (DCA) significantly influenced the growth of intestinal bacteria by impacting ribosome transcription and amino-acid metabolism. The metabolomic analysis underscores the pronounced impact of DCA on amino-acid profiles in both in vivo and in vitro settings. This study not only elucidates the effects of BAs on a diverse range of bacterial strains and their role in shaping the gut microbiota but also reveals underlying mechanisms essential for understanding and maintaining a healthy gut microbiota., (© 2024. The Author(s).)

5-fluorouracil (5-FU), a major anti-cancer therapeutic, is believed to function primarily by inhibiting thymidylate synthase, depleting deoxythymidine triphosphate (dTTP), and causing DNA damage. Here, we show that clinical combinations of 5-FU with oxaliplatin or irinotecan show no synergy in human colorectal cancer (CRC) trials and sub-additive killing in CRC cell lines. Using selective 5-FU metabolites, phospho- and ubiquitin proteomics, and primary human CRC organoids, we demonstrate that 5-FU-mediated CRC cell killing primarily involves an RNA damage response during ribosome biogenesis, causing lysosomal degradation of damaged rRNAs and proteasomal degradation of ubiquitinated ribosomal proteins. Tumor types clinically responsive to 5-FU treatment show upregulated rRNA biogenesis while 5-FU clinically non-responsive tumor types do not, instead showing greater sensitivity to 5-FU's DNA damage effects. Finally, we show that treatments upregulating ribosome biogenesis, including KDM2A inhibition, promote RNA-dependent cell killing by 5-FU, demonstrating the potential for combinatorial targeting of this ribosomal RNA damage response for improved cancer therapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Eukaryotic protein translation has slowly gained the scientific community's attention for its advanced and powerful therapeutic potential. However, recent technical developments in studying ribosomes and global translation have revolutionized our understanding of this complex multistep process. These developments have improved and deepened the current knowledge of mRNA translation, sparking excitement and new possibilities in this field. Translation factors are crucial for maintaining protein synthesis homeostasis. Since actively proliferating cancer cells depend on protein synthesis, dysregulated protein translation is central to tumorigenesis. Translation factors and their abnormal expressions directly affect multiple oncogenes and tumor suppressors. Recently, small molecules have been used to target translation factors, resulting in translation inhibition in a gene-specific manner, opening the door for developing translation inhibitors that can lead to novel chemotherapeutic drugs for treating multiple cancer types caused by dysregulated translation machinery. This review comprehensively summarizes the involvement of translation factors in tumor progression and oncogenesis. Also, it sheds light on the evolution of translation factors as novel drug targets for developing future therapeutic drugs for treating cancer.

Inhibitors of the integrated stress response (ISR) have been used to explore the potential beneficial effects of reducing the activation of this pathway in diseases. As the ISR is in essence a protective response, there is, however, a risk that inhibition may compromise the cell's ability to restore protein homeostasis. Here, we show that the experimental compound ISRIB impairs degradation of proteins by the ubiquitin-proteasome system (UPS) during proteotoxic stress in the cytosolic, but not nuclear, compartment. Accumulation of a UPS reporter substrate that is intercepted by ribosome quality control was comparable to the level observed after blocking the UPS with a proteasome inhibitor. Consistent with impairment of the cytosolic UPS, ISRIB treatment caused an accumulation of polyubiquitylated and detergent insoluble defective ribosome products (DRiPs) in the presence of puromycin. Our data suggest that the persistent protein translation during proteotoxic stress in the absence of a functional ISR increases the pool of DRiPs, thereby hindering the efficient clearance of cytosolic substrates by the UPS., (© 2024. The Author(s).)

Antibiotics targeting the bacterial ribosome are essential to combating bacterial infections. These antibiotics bind to various sites on the ribosome, inhibiting different stages of protein synthesis. This review provides a comprehensive overview of the mechanisms of action of clinically relevant antibiotics that target the bacterial ribosome, including macrolides, lincosamides, oxazolidinones, aminoglycosides, tetracyclines, and chloramphenicol. The structural and functional details of antibiotic interactions with ribosomal RNA, including specific binding sites, interactions with rRNA nucleotides, and their effects on translation processes, are discussed. Focus is placed on the diversity of these mechanisms and their clinical implications in treating bacterial infections, particularly in the context of emerging resistance. Understanding these mechanisms is crucial for developing novel therapeutic agents capable of overcoming bacterial resistance.

Background: Cannabidiol (CBD) is one of the main cannabinoids present in Cannabis sativa female flowers. Previous investigation has already provided insights into the CBD molecular mechanism; however, there is no transcriptome data for CBD effects on hippocampal subfields. Here, we investigate transcriptomic changes in dorsal and ventral CA1 of adult mice hippocampus after 100 mg/kg of CBD administration (i.p.) for one or seven consecutive days., Methods: C57BL/6JUnib mice were treated with either vehicle or CBD for 1 or 7 days. The collected brains were sectioned, and the hippocampal sub-regions were laser microdissected for RNA-Seq analysis., Results: The transcriptome analysis following 7 days of CBD administration indicates the differential expression of 1559 genes in dCA1 and 2924 genes in vCA1. Furthermore, GO/KEGG analysis identified 88 significantly enriched biological process and 26 significantly enriched pathways for dCBD7, whereas vCBD7 revealed 128 enriched BPs and 24 pathways., Conclusion: This dataset indicates a widespread decrease of electron transport chain and ribosome biogenesis transcripts in CA1, while chromatin modifications and synapse organization transcripts were increased following CBD administration for 7 days.

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., (© 2024. The Author(s).)

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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., Competing Interests: YB, YZ, YL, SL, RB, JS, YC, SW, DG No competing interests declared, (© 2023, Ban, Zou et al.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that the study was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., (© 2024. The Author(s).)

Endometrial carcinoma (ECa) is the fourth most common cancer among women. The oncogene PELP1 is frequently overexpressed in a variety of cancers, including ECa. We recently generated SMIP34, a small-molecule inhibitor of PELP1 that suppresses PELP1 oncogenic signaling. In this study, we assessed the effectiveness of SMIP34 in treating ECa. Treatment of established and primary patient-derived ECa cells with SMIP34 resulted in a significant reduction of cell viability, colony formation ability, and induction of apoptosis. RNA-seq analyses showed that SMIP34-regulated genes were negatively correlated with ribosome biogenesis and eukaryotic translation pathways. Mechanistic studies showed that the Rix complex, which is essential for ribosomal biogenesis, is disrupted upon SMIP34 binding to PELP1. Biochemical assays confirmed that SMIP34 reduced ribosomal biogenesis and new protein synthesis. Further, SMIP34 enhanced the efficacy of mTOR inhibitors in reducing viability of ECa cells. SMIP34 is also effective in reducing cell viability in ECa organoids in vitro and explants ex vivo. Importantly, SMIP34 treatment resulted in a significant reduction of the growth of ECa xenografts. Collectively, these findings underscore the potential of SMIP34 in treating ECa., (© 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

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., (© 2024. The Author(s).)

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.

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., (© 2024. The Author(s).)

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., (Copyright © 2024 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

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 RiboScreen TM , a technology for identifying molecules that enhance the production of specific proteins, to target the production of tropoelastin. We make use of RiboScreen TM 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 RiboScreen TM , 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 RiboScreen TM 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.

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., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Flentke et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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/β-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.

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., Competing Interests: GH, JW, KR, CJ, PP, TT, AF, LV, SL, BG, BS, MS, ER, SG, FM, QL No competing interests declared, MS Receives research funding from ALX Oncology, Astex, Incyte, Takeda and TG Therapeutics; has stock in Karyopharm and Ryvu; serves on advisory boards or consults for BMS, CTI, Forma, Geron, GSK, Karyopharm, Rigel, Ryvu, Taiho and Treadwell, TL, SF Patents: Lee T, Alvarado J, Tian J, Meyers KM, Han C, Mills JJ, Teuscher KB, Stauffer SR, Fesik SW. WDR5 inhibitors and modulators. WO 2020086857. 30 April 2020; Lee T, Han C, Mills JJ, Teuscher KB, Tian J, Meyers KM, Chowdhury S, Fesik SW. WDR5 inhibitors and modulators. WO 2020247679. 10 December 2020; Lee T, Teuscher KB, Tian J, Meyers KM, Chowdhury S, Fesik SW. WDR5 Inhibitors and modulators. WO 2021092525. 14 May 2021; Lee T, Teuscher KB, Chowdhury S, Tian J, Meyers KM, Fesik SW. WDR5 Inhibitors and modulators. WO2022236101. 10 November 2022, WT Patents: Fesik SW, Stauffer SR, Salovich JM, Tansey WP, Wang F, Phan J, Olejniczak ET, inventors. WDR5 inhibitors and modulators. United States Patent US 10,501,466. 10 December 2019; Fesik SW, Stauffer SR, Tansey WP, Olejniczak ET, Phan J, Wang F, Jeon K, Gogliotti RD, inventors. WDR5 inhibitors and modulators. United States Patent US 10,160,763. 25 December 2018, (© 2023, Howard et al.)

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.

Importance: Cfr is an antibiotic resistance enzyme that inhibits five clinically important antibiotic classes, is genetically mobile, and has a minimal fitness cost, making Cfr a serious threat to antibiotic efficacy. The significance of our work is in discovering molecules that inhibit Cfr-dependent methylation of the ribosome, thus protecting the efficacy of the PhLOPS A antibiotics. These molecules are the first reported inhibitors of Cfr-mediated ribosome methylation and, as such, will guide the further discovery of chemical scaffolds against Cfr-mediated antibiotic resistance. Our work acts as a foundation for further development of molecules that safeguard the PhLOPS A antibiotics from Cfr., Competing Interests: The authors declare no conflict of interest.

Acne vulgaris is a chronic disfiguring skin disease affecting ∼1 billion people worldwide, often having persistent negative effects on physical and mental health. The Gram-positive anaerobe, Cutibacterium acnes is implicated in acne pathogenesis and is, therefore, a main target for antibiotic-based acne therapy. We determined a 2.8-Å resolution structure of the 70S ribosome of Cutibacterium acnes by cryogenic electron microscopy and discovered that sarecycline, a narrow-spectrum antibiotic against Cutibacterium acnes, may inhibit two active sites of this bacterium's ribosome in contrast to the one site detected previously on the model ribosome of Thermus thermophilus. Apart from the canonical binding site at the mRNA decoding center, the second binding site for sarecycline exists at the nascent peptide exit tunnel, reminiscent of the macrolides class of antibiotics. The structure also revealed Cutibacterium acnes-specific features of the ribosomal RNA and proteins. Unlike the ribosome of the Gram-negative bacterium Escherichia coli, Cutibacterium acnes ribosome has two additional proteins, bS22 and bL37, which are also present in the ribosomes of Mycobacterium smegmatis and Mycobacterium tuberculosis. We show that bS22 and bL37 have antimicrobial properties and may be involved in maintaining the healthy homeostasis of the human skin microbiome., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells 1 . Here we use advances in cryo-electron tomography and sub-tomogram analysis 2,3 to visualize the structural dynamics of translation inside the bacterium Mycoplasma pneumoniae. To interpret the functional states in detail, we first obtain a high-resolution in-cell average map of all translating ribosomes and build an atomic model for the M. pneumoniae ribosome that reveals distinct extensions of ribosomal proteins. Classification then resolves 13 ribosome states that differ in their conformation and composition. These recapitulate major states that were previously resolved in vitro, and reflect intermediates during active translation. On the basis of these states, we animate translation elongation inside native cells and show how antibiotics reshape the cellular translation landscapes. During translation elongation, ribosomes often assemble in defined three-dimensional arrangements to form polysomes 4 . By mapping the intracellular organization of translating ribosomes, we show that their association into polysomes involves a local coordination mechanism that is mediated by the ribosomal protein L9. We propose that an extended conformation of L9 within polysomes mitigates collisions to facilitate translation fidelity. Our work thus demonstrates the feasibility of visualizing molecular processes at atomic detail inside cells., (© 2022. The Author(s).)

Human NLRP1 (NACHT, LRR, and PYD domain-containing protein 1) is an innate immune sensor predominantly expressed in the skin and airway epithelium. Here, we report that human NLRP1 senses the ultraviolet B (UVB)- and toxin-induced ribotoxic stress response (RSR). Biochemically, RSR leads to the direct hyperphosphorylation of a human-specific disordered linker region of NLRP1 (NLRP1 DR ) by MAP3K20/ZAKα kinase and its downstream effector, p38. Mutating a single ZAKα phosphorylation site in NLRP1 DR abrogates UVB- and ribotoxin-driven pyroptosis in human keratinocytes. Moreover, fusing NLRP1 DR to CARD8, which is insensitive to RSR by itself, creates a minimal inflammasome sensor for UVB and ribotoxins. These results provide insight into UVB sensing by human skin keratinocytes, identify several ribotoxins as NLRP1 agonists, and establish inflammasome-driven pyroptosis as an integral component of the RSR.

Ribosome-targeting antibiotics serve as powerful antimicrobials and as tools for studying the ribosome, the catalytic peptidyl transferase center (PTC) of which is targeted by many drugs. The classic PTC-acting antibiotic chloramphenicol (CHL) and the newest clinically significant linezolid (LZD) were considered indiscriminate inhibitors of protein synthesis that cause ribosome stalling at every codon of every gene being translated. However, recent discoveries have shown that CHL and LZD preferentially arrest translation when the ribosome needs to polymerize particular amino acid sequences. The molecular mechanisms that underlie the context-specific action of ribosome inhibitors are unknown. Here we present high-resolution structures of ribosomal complexes, with or without CHL, carrying specific nascent peptides that support or negate the drug action. Our data suggest that the penultimate residue of the nascent peptide directly modulates antibiotic affinity to the ribosome by either establishing specific interactions with the drug or by obstructing its proper placement in the binding site., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The antibiotic linezolid, the first clinically approved member of the oxazolidinone class, inhibits translation of bacterial ribosomes by binding to the peptidyl transferase center. Recent work has demonstrated that linezolid does not inhibit peptide bond formation at all sequences but rather acts in a context-specific manner, namely when alanine occupies the penultimate position of the nascent chain. However, the molecular basis for context-specificity has not been elucidated. Here we show that the second-generation oxazolidinone radezolid also induces stalling with a penultimate alanine, and we determine high-resolution cryo-EM structures of linezolid- and radezolid-stalled ribosome complexes to explain their mechanism of action. These structures reveal that the alanine side chain fits within a small hydrophobic crevice created by oxazolidinone, resulting in improved ribosome binding. Modification of the ribosome by the antibiotic resistance enzyme Cfr disrupts stalling due to repositioning of the modified nucleotide. Together, our findings provide molecular understanding for the context-specificity of oxazolidinones., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Actinomycin D has been reported to selectively inhibit rRNA synthesis and ribosome biogenesis, induce G2 checkpoint of cell cycle arrest in HeLa cells. In Arabidopsis, actinomycin D was also used as agent to preferentially inhibit the ribosome biosynthesis and ribosomal function. However, the function of actinomycin D on Arabidopsis root development remains to be elucidated. In this study, we exposed Arabidopsis seedlings to actinomycin D with the aim of evaluating the effects of ribosome biogenesis on root development. The results demonstrated that actinomycin D inhibited Arabidopsis root growth by reduced meristematic activity in a dose dependent manner. Exposure to actinomycin D decreased the expression of WOX5 and key stem cell niche-defining transcription factors SHR and PLT1, thus the loss function of QC identity and stem cell niche maintenance. In addition, dead cells were observed after actinomycin D treatment in root stele initials and DNA damage response was constitutively activated. Collectively, we propose that ribosome biogenesis plays key role in primary root growth through maintenance of root stem cell niche and DNA damage response in Arabidopsis., Competing Interests: Declaration of competing interest The authors declared that they have no conflicts of interest to this work., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Mechanisms of drug-tolerance remain poorly understood and have been linked to genomic but also to non-genomic processes. 5-fluorouracil (5-FU), the most widely used chemotherapy in oncology is associated with resistance. While prescribed as an inhibitor of DNA replication, 5-FU alters all RNA pathways. Here, we show that 5-FU treatment leads to the production of fluorinated ribosomes exhibiting altered translational activities. 5-FU is incorporated into ribosomal RNAs of mature ribosomes in cancer cell lines, colorectal xenografts, and human tumors. Fluorinated ribosomes appear to be functional, yet, they display a selective translational activity towards mRNAs depending on the nature of their 5'-untranslated region. As a result, we find that sustained translation of IGF-1R mRNA, which encodes one of the most potent cell survival effectors, promotes the survival of 5-FU-treated colorectal cancer cells. Altogether, our results demonstrate that "man-made" fluorinated ribosomes favor the drug-tolerant cellular phenotype by promoting translation of survival genes., (© 2022. The Author(s).)

Chemotherapeutic agents (CAs) are first-line antineoplastic treatments against a wide variety of cancers. Despite their effectiveness in halting tumor progression, side effects associated with CAs promote muscle loss by incompletely understood mechanisms. To address this problem, we first identified how oxidative stress impairs protein synthesis in C2C12 myotubes. Transient elevations in reactive oxygen species (ROS) resulted in protein synthesis deficits and reduced ribosomal (r)RNA levels. Oxidative stress did not reduce rRNA gene (rDNA) transcription, but it caused an increase in rRNA and protein oxidation. To determine whether CAs affect protein synthesis independent of oxidative stress, we exposed myotubes to Paclitaxel (PTX), Doxorubicin (DXR), or Marizomib (Mzb) at doses that did result in elevated ROS levels (sub-ROS). Exposure to CAs reduced protein synthesis and rRNA levels, but unlike oxidative stress, sub-ROS exposures impaired rDNA transcription. These results indicate that although oxidative stress disrupts protein synthesis by compromising ribosomal quantity and quality, CAs at sub-ROS doses compromise protein synthesis and ribosomal capacity, at least in part, by reducing rDNA transcription. Therefore, CAs negatively impact protein synthesis by causing oxidative stress in addition to directly reducing the ribosomal capacity of myotubes in a ROS-independent manner.

Guanine quadruplexes (G4s) are non-canonical nucleic acid structures commonly found in regulatory genomic regions. G4 targeting has emerged as a therapeutic approach in cancer. We have screened naphthalene-diimides (NDIs), a class of G4 ligands, in a cellular model of colorectal cancer (CRC). Here, we identify the leading compound T5 with a potent and selective inhibition of cell growth by high-affinity binding to G4s in ribosomal DNA, impairing RNA polymerase I (Pol I) elongation. Consequently, T5 induces a rapid inhibition of Pol I transcription, nucleolus disruption, proteasome-dependent Pol I catalytic subunit A degradation and autophagy. Moreover, we attribute the higher selectivity of carbohydrate-conjugated T5 for tumoral cells to its preferential uptake through the overexpressed glucose transporter 1. Finally, we succinctly demonstrate that T5 could be explored as a therapeutic agent in a patient cohort with CRC. Therefore, we report a mode of action for these NDIs involving ribosomal G4 targeting., Competing Interests: Declaration of interests The authors declare no competing interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The Saccharomyces cerevisiae gene deletion collection is widely used for functional gene annotation and genetic interaction analyses. However, the standard G418-resistance cassette used to produce knockout mutants delivers strong regulatory elements into the target genetic loci. To date, its side effects on the expression of neighboring genes have never been systematically assessed. Here, using ribosome profiling data, RT-qPCR, and reporter expression, we investigated perturbations induced by the KanMX module. Our analysis revealed significant alterations in the transcription efficiency of neighboring genes and, more importantly, severe impairment of their mRNA translation, leading to changes in protein abundance. In the 'head-to-head' orientation of the deleted and neighboring genes, knockout often led to a shift of the transcription start site of the latter, introducing new uAUG codon(s) into the expanded 5' untranslated region (5' UTR). In the 'tail-to-tail' arrangement, knockout led to activation of alternative polyadenylation signals in the neighboring gene, thus altering its 3' UTR. These events may explain the so-called neighboring gene effect (NGE), i.e. false genetic interactions of the deleted genes. We estimate that in as much as ∼1/5 of knockout strains the expression of neighboring genes may be substantially (>2-fold) deregulated at the level of translation., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The dearth of new medicines effective against antibiotic-resistant bacteria presents a growing global public health concern 1 . For more than five decades, the search for new antibiotics has relied heavily on the chemical modification of natural products (semisynthesis), a method ill-equipped to combat rapidly evolving resistance threats. Semisynthetic modifications are typically of limited scope within polyfunctional antibiotics, usually increase molecular weight, and seldom permit modifications of the underlying scaffold. When properly designed, fully synthetic routes can easily address these shortcomings 2 . Here we report the structure-guided design and component-based synthesis of a rigid oxepanoproline scaffold which, when linked to the aminooctose residue of clindamycin, produces an antibiotic of exceptional potency and spectrum of activity, which we name iboxamycin. Iboxamycin is effective against ESKAPE pathogens including strains expressing Erm and Cfr ribosomal RNA methyltransferase enzymes, products of genes that confer resistance to all clinically relevant antibiotics targeting the large ribosomal subunit, namely macrolides, lincosamides, phenicols, oxazolidinones, pleuromutilins and streptogramins. X-ray crystallographic studies of iboxamycin in complex with the native bacterial ribosome, as well as with the Erm-methylated ribosome, uncover the structural basis for this enhanced activity, including a displacement of the [Formula: see text] nucleotide upon antibiotic binding. Iboxamycin is orally bioavailable, safe and effective in treating both Gram-positive and Gram-negative bacterial infections in mice, attesting to the capacity for chemical synthesis to provide new antibiotics in an era of increasing resistance., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Ricin toxin A subunit (RTA) is the catalytic subunit of ricin, which depurinates an adenine from the sarcin/ricin loop in eukaryotic ribosomes. There are no approved inhibitors against ricin. We used a new strategy to disrupt RTA-ribosome interactions by fragment screening using surface plasmon resonance. Here, using a structure-guided approach, we improved the affinity and inhibitory activity of small-molecular-weight lead compounds and obtained improved compounds with over an order of magnitude higher efficiency. Four advanced compounds were characterized by X-ray crystallography. They bind at the RTA-ribosome binding site as the original compound but in a distinctive manner. These inhibitors bind remotely from the catalytic site and cause local conformational changes with no alteration of the catalytic site geometry. Yet they inhibit depurination by ricin holotoxin and inhibit the cytotoxicity of ricin in mammalian cells. They are the first agents that protect against ricin holotoxin by acting directly on RTA.

The anticancer effect of zinc oxide nanoparticles (ZnO NPs) largely relies on cellular responses such as alteration of gene expression. Although ZnO NPs have been reported to induce transcriptional changes, the potential of ZnO NPs to affect cellular translatome remains largely unknown. Using ribosome profiling, we demonstrated that the transcription of 78 genes and the translation of 1,448 genes are affected during one hour of ZnO NPs exposure in A549 human lung cancer cells. The mitogen-activated protein kinase (MAPK) pathway is up-regulated upon ZnO NP treatment. The upstream open reading frame (uORF) plays a pervasive role in the induction of up-regulated genes, including TLNRD1 and CCNB1IP1 . Knockdown of TLNRD1 or CCNB1IP1 reduces ZnO NP-induced cytotoxicity. Together, our study characterizes the landscape of translational alteration under ZnO NPs treatment and provides potential targets to augment the anticancer effect of ZnO NPs.

Shiga toxins (Stxs), also known as Shiga-like toxins (SLT) or verotoxins (VT), constitute a family of structurally and functionally related cytotoxic proteins produced by the enteric pathogens Shigella dysenteriae type 1 and Stx-producing Escherichia coli (STEC). Infection with these bacteria causes bloody diarrhea and other pathological manifestations that can lead to HUS (hemolytic and uremic syndrome). At the cellular level, Stxs bind to the cellular receptor Gb3 and inhibit protein synthesis by removing an adenine from the 28S rRNA. This triggers multiple cellular signaling pathways, including the ribotoxic stress response (RSR), unfolded protein response (UPR), autophagy and apoptosis. Stxs cause several pathologies of major public health concern, but their specific targeting of host cells and efficient delivery to the cytosol could potentially be exploited for biomedical purposes. Moreover, high levels of expression have been reported for the Stxs receptor, Gb3/CD77, in Burkitt's lymphoma (BL) cells and on various types of solid tumors. These properties have led to many attempts to develop Stxs as tools for biomedical applications, such as cancer treatment or imaging, and several engineered Stxs are currently being tested. We provide here an overview of these studies.

In the traditional fermentative model yeast Saccharomyces cerevisiae , Sc Ixr1 is an HMGB (High Mobility Group box B) protein that has been considered as an important regulator of gene transcription in response to external changes like oxygen, carbon source, or nutrient availability. Kluyveromyces lactis is also a useful eukaryotic model, more similar to many human cells due to its respiratory metabolism. We cloned and functionally characterized by different methodologies Kl IXR1, which encodes a protein with only 34.4% amino acid sequence similarity to Sc Ixr1. Our data indicate that both proteins share common functions, including their involvement in the response to hypoxia or oxidative stress induced by hydrogen peroxide or metal treatments, as well as in the control of key regulators for maintenance of the dNTP (deoxyribonucleotide triphosphate) pool and ribosome synthesis. Kl Ixr1 is able to bind specific regulatory DNA sequences in the promoter of its target genes, which are well conserved between S. cerevisiae and K. lactis . Oppositely, we found important differences between Sc Irx1 and Kl Ixr1 affecting cellular responses to cisplatin or cycloheximide in these yeasts, which could be dependent on specific and non-conserved domains present in these two proteins.

Macrolides have been effective clinical antibiotics for over 70 years. They inhibit protein biosynthesis in bacterial pathogens by narrowing the nascent protein exit tunnel in the ribosome. The macrolide class of natural products consist of a macrolactone ring linked to one or more sugar molecules. Most of the macrolides used currently are semi-synthetic erythromycin derivatives, composed of a 14- or 15-membered macrolactone ring. Rapidly emerging resistance in bacterial pathogens is among the most urgent global health challenges, which render many antibiotics ineffective, including next-generation macrolides. To address this threat and advance a longer-term plan for developing new antibiotics, we demonstrate how 16-membered macrolides overcome erythromycin resistance in clinically isolated Staphylococcus aureus strains. By determining the structures of complexes of the large ribosomal subunit of Deinococcus radiodurans (D50S) with these 16-membered selected macrolides, and performing anti-microbial studies, we identified resistance mechanisms they may overcome. This new information provides important insights toward the rational design of therapeutics that are effective against drug resistant human pathogens., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Single-cell sequencing methods have enabled in-depth analysis of the diversity of cell types and cell states in a wide range of organisms. These tools focus predominantly on sequencing the genomes 1 , epigenomes 2 and transcriptomes 3 of single cells. However, despite recent progress in detecting proteins by mass spectrometry with single-cell resolution 4 , it remains a major challenge to measure translation in individual cells. Here, building on existing protocols 5-7 , we have substantially increased the sensitivity of these assays to enable ribosome profiling in single cells. Integrated with a machine learning approach, this technology achieves single-codon resolution. We validate this method by demonstrating that limitation for a particular amino acid causes ribosome pausing at a subset of the codons encoding the amino acid. Of note, this pausing is only observed in a sub-population of cells correlating to its cell cycle state. We further expand on this phenomenon in non-limiting conditions and detect pronounced GAA pausing during mitosis. Finally, we demonstrate the applicability of this technique to rare primary enteroendocrine cells. This technology provides a first step towards determining the contribution of the translational process to the remarkable diversity between seemingly identical cells., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

In general, mRNAs are assumed to be loaded with ribosomes instantly upon entry into the cytoplasm. To measure ribosome density (RD) on nascent mRNA, we developed nascent Ribo-Seq by combining Ribo-Seq with progressive 4-thiouridine labeling. In mouse macrophages, we determined experimentally the lag between the appearance of nascent mRNA and its association with ribosomes, which was calculated to be 20-22 min for bulk mRNA. In mouse embryonic stem cells, nRibo-Seq revealed an even stronger lag of 35-38 min in ribosome loading. After stimulation of macrophages with lipopolysaccharide, the lag between cytoplasmic and translated mRNA leads to uncoupling between input and ribosome-protected fragments, which gives rise to distorted RD measurements under conditions where mRNA amounts are far from steady-state expression. As a result, we demonstrate that transcriptional changes affect RD in a passive way., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Ribosome profiling measures genome-wide translation dynamics at sub-codon resolution. Cycloheximide (CHX), a widely used translation inhibitor to arrest ribosomes in these experiments, has been shown to induce biases in yeast, questioning its use. However, whether such biases are present in datasets of other organisms including humans is unknown. Here we compare different CHX-treatment conditions in human cells and yeast in parallel experiments using an optimized protocol. We find that human ribosomes are not susceptible to conformational restrictions by CHX, nor does it distort gene-level measurements of ribosome occupancy, measured decoding speed or the translational ramp. Furthermore, CHX-induced codon-specific biases on ribosome occupancy are not detectable in human cells or other model organisms. This shows that reported biases of CHX are species-specific and that CHX does not affect the outcome of ribosome profiling experiments in most settings. Our findings provide a solid framework to conduct and analyze ribosome profiling experiments., (© 2021. The Author(s).)

Eukaryotic protein synthesis is the highly conserved, complex mechanism of translating genetic information into proteins. Although this process is essential for cellular homoeostasis, dysregulations are associated with cellular malfunctions and diseases including cancer and diabetes. In the challenging and ongoing search for adequate treatment possibilities, natural products represent excellent research tools and drug leads for new interactions with the translational machinery and for influencing mRNA translation. In this review, bacterial-, marine- and plant-derived natural compounds that interact with different steps of mRNA translation, comprising ribosomal assembly, translation initiation and elongation, are highlighted. Thereby, the exact binding and interacting partners are unveiled in order to accurately understand the mode of action of each natural product. The pharmacological relevance of these compounds is furthermore assessed by evaluating the observed biological activities in the light of translational inhibition and by enlightening potential obstacles and undesired side-effects, e.g. in clinical trials. As many of the natural products presented here possess the potential to serve as drug leads for synthetic derivatives, structural motifs, which are indispensable for both mode of action and biological activities, are discussed. Evaluating the natural products emphasises the strong diversity of their points of attack. Especially the fact that selected binding partners can be set in direct relation to different diseases emphasises the indispensability of natural products in the field of drug development. Discovery of new, unique and unusual interacting partners again renders them promising tools for future research in the field of eukaryotic mRNA translation., (Copyright © 2021 Elsevier Ltd. All rights reserved.)