Integrated ribosome and proteome analyses reveal insights into sevoflurane-induced long-term social behavior and cognitive dysfunctions through ADNP inhibition in neonatal mice.

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.

Antimicrobial macrozones interact with biological macromolecules via two-site binding mode of action: Fluorimetric, NMR and docking studies.

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.

Edodin: A New Type of Toxin from Shiitake Mushroom (Lentinula edodes) That Inactivates Mammalian Ribosomes.

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.

Ribosome subunit attrition and activation of the p53-MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition.

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.

Sarecycline inhibits protein translation in Cutibacterium acnes 70S ribosome using a two-site mechanism.

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-Šresolution 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.

Visualizing translation dynamics at atomic detail inside a bacterial cell.

Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells1. Here we use advances in cryo-electron tomography and sub-tomogram analysis2,3 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 polysomes4. 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.

ZAKα-driven ribotoxic stress response activates the human NLRP1 inflammasome.

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 (NLRP1DR) by MAP3K20/ZAKα kinase and its downstream effector, p38. Mutating a single ZAKα phosphorylation site in NLRP1DR abrogates UVB- and ribotoxin-driven pyroptosis in human keratinocytes. Moreover, fusing NLRP1DR 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.

Structural basis for context-specific inhibition of translation by oxazolidinone antibiotics.

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.

Structural basis for the context-specific action of the classic peptidyl transferase inhibitor chloramphenicol.

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.

Alteration of ribosome function upon 5-fluorouracil treatment favors cancer cell drug-tolerance.

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.

Inhibition of ribosome biogenesis by actinomycin D affects Arabidopsis root development.

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.

A standard knockout procedure alters expression of adjacent loci at the translational level.

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.

Ribosome profiling reveals translatome remodeling in cancer cells in response to zinc oxide nanoparticles.

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 as Antitumor Tools.

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.

Synthesis and Structural Characterization of Ricin Inhibitors Targeting Ribosome Binding Using Fragment-Based Methods and Structure-Based Design.

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.

Chemotherapy agents reduce protein synthesis and ribosomal capacity in myotubes independent of oxidative stress.

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.

A synthetic antibiotic class overcoming bacterial multidrug resistance.

The dearth of new medicines effective against antibiotic-resistant bacteria presents a growing global public health concern1. 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 shortcomings2. 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.

Single-cell Ribo-seq reveals cell cycle-dependent translational pausing.

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 genomes1, epigenomes2 and transcriptomes3 of single cells. However, despite recent progress in detecting proteins by mass spectrometry with single-cell resolution4, it remains a major challenge to measure translation in individual cells. Here, building on existing protocols5-7, 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.

The HMGB Protein KlIxr1, a DNA Binding Regulator of Kluyveromyces lactis Gene Expression Involved in Oxidative Metabolism, Growth, and dNTP Synthesis.

In the traditional fermentative model yeast Saccharomyces cerevisiae, ScIxr1 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 KlIXR1, which encodes a protein with only 34.4% amino acid sequence similarity to ScIxr1. 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. KlIxr1 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 ScIrx1 and KlIxr1 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.