First-in-Class Inhibitors of the Ribosomal Oxygenase MINA53.

MINA53 is a JmjC domain 2-oxoglutarate-dependent oxygenase that catalyzes ribosomal hydroxylation and is a target of the oncogenic transcription factor c-MYC. Despite its anticancer target potential, no small-molecule MINA53 inhibitors are reported. Using ribosomal substrate fragments, we developed mass spectrometry assays for MINA53 and the related oxygenase NO66. These assays enabled the identification of 2-(aryl)alkylthio-3,4-dihydro-4-oxoypyrimidine-5-carboxylic acids as potent MINA53 inhibitors, with selectivity over NO66 and other JmjC oxygenases. Crystallographic studies with the JmjC demethylase KDM5B revealed active site binding but without direct metal chelation; however, molecular modeling investigations indicated that the inhibitors bind to MINA53 by directly interacting with the iron cofactor. The MINA53 inhibitors manifest evidence for target engagement and selectivity for MINA53 over KDM4-6. The MINA53 inhibitors show antiproliferative activity with solid cancer lines and sensitize cancer cells to conventional chemotherapy, suggesting that further work investigating their potential in combination therapies is warranted.

Engineering the Translational Machinery for Biotechnology Applications.

The ribosome is an essential organelle in charge of the translational processes in all kinds of cells. Currently, the scenario of its function has been significantly expanded from the classic machine for protein synthesis to a regulatory platform for quality control to maintain the protein homeostasis in a living cell. The ribosome is much more than a mechanical device with a static structure: it is inherently dynamic in structure and function, especially in response to the environmental fluctuations. Considerable effort has been made to regulate its structure and physiological function by engineering the components of a ribosome. The findings of the pioneering studies significantly deepened our understanding of a ribosome and exemplified how a ribosome could be engineered for biotechnology purposes in the era of synthetic biology. The engineering of ribosome offered highly accessible methods capable of comprehensively optimizing the performance of strains of industrial importance. In this article, the relevant recent advances were systematically reviewed.

Discovery of Functional Macrocyclic Peptides by Means of the RaPID System.

Flexizymes, highly flexible tRNA aminoacylation ribozymes, have enabled charging of virtually any amino acid (including non-proteogenic ones) onto tRNA molecules. Coupling to a custom-made in vitro translation system, namely the flexible in vitro translation (FIT) system, has unveiled the remarkable tolerance of the ribosome toward molecules, remote from what nature has selected to carry out its elaborate functions. Among the very diverse molecules and chemistries that have been ribosomally incorporated, a plethora of entities capable of mediating intramolecular cyclization have revolutionized the design and discovery of macrocyclic peptides. These macrocyclization reactions (which can be spontaneous, chemical, or enzymatic) have all served as tools for the discovery of peptides with natural-like structures and properties. Coupling of the FIT system and mRNA display techniques, known as the random non-standard peptide integrated discovery (RaPID) system, has in turn allowed for the simultaneous screening of trillions of macrocyclic peptides against challenging biological targets. The macrocyclization methodologies are chosen depending on the structural and functional characteristics of the desired molecule. Thus, they can emanate from the peptide's N-terminus or its side chains, attributing flexibility or rigidity, or even result in the installation of fluorescent probes.

A tandem active site model for the ribosomal helicase.

During protein synthesis, the messenger RNA (mRNA) helicase activity of the ribosome ensures that codons are made single stranded before decoding. Here, based on recent structural and functional findings, a quantitative model is presented for a tandem arrangement of two helicase active sites on the ribosome. A distal site encounters mRNA structures first, one elongation cycle earlier than a proximal site. Although unwinding of encountered mRNA structures past the proximal site is required for translocation, two routes exist for translocation past the distal site: sliding, which requires unwinding, and stick-slip, which does not. The model accounts in detail for a number of findings related to the ribosomal helicase and provides a testable framework to further study mRNA unwinding.

Architecture of Microcin B17 Synthetase: An Octameric Protein Complex Converting a Ribosomally Synthesized Peptide into a DNA Gyrase Poison.

The introduction of azole heterocycles into a peptide backbone is the principal step in the biosynthesis of numerous compounds with therapeutic potential. One of them is microcin B17, a bacterial topoisomerase inhibitor whose activity depends on the conversion of selected serine and cysteine residues of the precursor peptide to oxazoles and thiazoles by the McbBCD synthetase complex. Crystal structures of McbBCD reveal an octameric B4C2D2 complex with two bound substrate peptides. Each McbB dimer clamps the N-terminal recognition sequence, while the C-terminal heterocycle of the modified peptide is trapped in the active site of McbC. The McbD and McbC active sites are distant from each other, which necessitates alternate shuttling of the peptide substrate between them, while remaining tethered to the McbB dimer. An atomic-level view of the azole synthetase is a starting point for deeper understanding and control of biosynthesis of a large group of ribosomally synthesized natural products.

Deciphering Determinants in Ribosomal Methyltransferases That Confer Antimicrobial Resistance.

Post-translational methylation of rRNA at select positions is a prevalent resistance mechanism adopted by pathogens. In this work, KsgA, a housekeeping ribosomal methyltransferase (rMtase) involved in ribosome biogenesis, was exploited as a model system to delineate the specific targeting determinants that impart substrate specificity to rMtases. With a combination of evolutionary and structure-guided approaches, a set of chimeras were created that altered the targeting specificity of KsgA such that it acted similarly to erythromycin-resistant methyltransferases (Erms), rMtases found in multidrug-resistant pathogens. The results revealed that specific loop embellishments on the basic Rossmann fold are key determinants in the selection of the cognate RNA. Moreover, in vivo studies confirmed that chimeric constructs are competent in imparting macrolide resistance. This work explores the factors that govern the emergence of resistance and paves the way for the design of specific inhibitors useful in reversing antibiotic resistance.

The emerging roles of ribosomal histidyl hydroxylases in cell biology, physiology and disease.

Hydroxylation is a novel protein modification catalyzed by a family of oxygenases that depend on fundamental nutrients and metabolites for activity. Protein hydroxylases have been implicated in a variety of key cellular processes that play important roles in both normal homeostasis and pathogenesis. Here, in this review, we summarize the current literature on a highly conserved sub-family of oxygenases that catalyze protein histidyl hydroxylation. We discuss the evidence supporting the biochemical assignment of these emerging enzymes as ribosomal protein hydroxylases, and provide an overview of their role in immunology, bone development, and cancer.

Patient-derived Models of Abiraterone- and Enzalutamide-resistant Prostate Cancer Reveal Sensitivity to Ribosome-directed Therapy.

BACKGROUND: The intractability of castration-resistant prostate cancer (CRPC) is exacerbated by tumour heterogeneity, including diverse alterations to the androgen receptor (AR) axis and AR-independent phenotypes. The availability of additional models encompassing this heterogeneity would facilitate the identification of more effective therapies for CRPC. OBJECTIVE: To discover therapeutic strategies by exploiting patient-derived models that exemplify the heterogeneity of CRPC. DESIGN, SETTING, AND PARTICIPANTS: Four new patient-derived xenografts (PDXs) were established from independent metastases of two patients and characterised using integrative genomics. A panel of rationally selected drugs was tested using an innovative ex vivo PDX culture system. INTERVENTION: The following drugs were evaluated: AR signalling inhibitors (enzalutamide and galeterone), a PARP inhibitor (talazoparib), a chemotherapeutic (cisplatin), a CDK4/6 inhibitor (ribociclib), bromodomain and extraterminal (BET) protein inhibitors (iBET151 and JQ1), and inhibitors of ribosome biogenesis/function (RNA polymerase I inhibitor CX-5461 and pan-PIM kinase inhibitor CX-6258). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Drug efficacy in ex vivo cultures of PDX tissues was evaluated using immunohistochemistry for Ki67 and cleaved caspase-3 levels. Candidate drugs were also tested for antitumour efficacy in vivo, with tumour volume being the primary endpoint. Two-tailed t tests were used to compare drug and control treatments. RESULTS AND LIMITATIONS: Integrative genomics revealed that the new PDXs exhibited heterogeneous mechanisms of resistance, including known and novel AR mutations, genomic structural rearrangements of the AR gene, and a neuroendocrine-like AR-null phenotype. Despite their heterogeneity, all models were sensitive to the combination of ribosome-targeting agents CX-5461 and CX-6258. CONCLUSIONS: This study demonstrates that ribosome-targeting drugs may be effective against diverse CRPC subtypes including AR-null disease, and highlights the potential of contemporary patient-derived models to prioritise treatment strategies for clinical translation. PATIENT SUMMARY: Diverse types of therapy-resistant prostate cancers are sensitive to a new combination of drugs that inhibit protein synthesis pathways in cancer cells.

The universally conserved GTPase HflX is an RNA helicase that restores heat-damaged Escherichia coli ribosomes.

The ribosome-associated GTPase HflX acts as an antiassociation factor upon binding to the 50S ribosomal subunit during heat stress in Escherichia coli Although HflX is recognized as a guanosine triphosphatase, several studies have shown that the N-terminal domain 1 of HflX is capable of hydrolyzing adenosine triphosphate (ATP), but the functional role of its adenosine triphosphatase (ATPase) activity remains unknown. We demonstrate that E. coli HflX possesses ATP-dependent RNA helicase activity and is capable of unwinding large subunit ribosomal RNA. A cryo-electron microscopy structure of the 50S-HflX complex in the presence of nonhydrolyzable analogues of ATP and guanosine triphosphate hints at a mode of action for the RNA helicase and suggests the linker helical domain may have a determinant role in RNA unwinding. Heat stress results in inactivation of the ribosome, and we show that HflX can restore heat-damaged ribosomes and improve cell survival.

Amazing Diversity in Biochemical Roles of Fe(II)/2-Oxoglutarate Oxygenases.

Since their discovery in the 1960s, the family of Fe(II)/2-oxoglutarate-dependent oxygenases has undergone a tremendous expansion to include enzymes catalyzing a vast diversity of biologically important reactions. Recent examples highlight roles in controlling chromatin modification, transcription, mRNA demethylation, and mRNA splicing. Others generate modifications in tRNA, translation factors, ribosomes, and other proteins. Thus, oxygenases affect all components of molecular biology's central dogma, in which information flows from DNA to RNA to proteins. These enzymes also function in biosynthesis and catabolism of cellular metabolites, including antibiotics and signaling molecules. Due to their critical importance, ongoing efforts have targeted family members for the development of specific therapeutics. This review provides a general overview of recently characterized oxygenase reactions and their key biological roles.

Activation of the Stringent Response by Loading of RelA-tRNA Complexes at the Ribosomal A-Site.

RelA/SpoT homologs (RSHs) are ubiquitous bacterial enzymes that synthesize and hydrolyze (p)ppGpp in response to environmental challenges. Bacteria cannot survive in hosts and produce infection without activating the (p)ppGpp-mediated stringent response, but it is not yet understood how the enzymatic activities of RSHs are controlled. Using UV crosslinking and deep sequencing, we show that Escherichia coli RelA ((p)ppGpp synthetase I) interacts with uncharged tRNA without being activated. Amino acid starvation leads to loading of cognate tRNA⋅RelA complexes at vacant ribosomal A-sites. In turn, RelA is activated and synthesizes (p)ppGpp. Mutation of a single, conserved residue in RelA simultaneously prevents tRNA binding, ribosome binding, and activation of RelA, showing that all three processes are interdependent. Our results support a model in which (p)ppGpp synthesis occurs by ribosome-bound RelA interacting with the Sarcin-Ricin loop of 23S rRNA.

Distribution of the ribosome associated endonuclease Rae1 and the potential role of conserved amino acids in codon recognition.

We recently identified a novel ribonuclease in Bacillus subtilis called Rae1 that cleaves mRNAs in a translation-dependent manner. Rae1 is a member of the NYN/PIN family of ribonucleases and is highly conserved in the Firmicutes, the Cyanobacteria and the chloroplasts of photosynthetic algae and plants. We have proposed a model in which Rae1 enters the A-site of ribosomes that are paused following translation of certain sequences that are still ill-defined. In the only case identified thus far, Rae1 cleaves between a conserved glutamate and lysine codon during translation of a short peptide called S1025. Certain other codons are also tolerated on either side of the cleavage site, but these are recognized less efficiently. The model of Rae1 docked in the A-site allows us to make predictions about which conserved residues may be important for recognition of mRNA, the tRNA in the adjacent P-site and binding to the 50S ribosome subunit.

Purification and characterization of Arabidopsis thaliana oligosaccharyltransferase complexes from the native host: a protein super-expression system for structural studies.

The oligosaccharyltransferase (OT) complex catalyzes N-glycosylation of nascent secretory polypeptides in the lumen of the endoplasmic reticulum. Despite their importance, little is known about the structure and function of plant OT complexes, mainly due to lack of efficient recombinant protein production systems suitable for studies on large plant protein complexes. Here, we purified Arabidopsis OT complexes using the tandem affinity-tagged OT subunit STAUROSPORINE AND TEMPERATURE SENSITIVE3a (STT3a) expressed by an Arabidopsis protein super-expression platform. Mass-spectrometry analysis of the purified complexes identified three essential OT subunits, OLIGOSACCHARYLTRANSFERASE1 (OST1), HAPLESS6 (HAP6), DEFECTIVE GLYCOSYLATION1 (DGL1), and a number of ribosomal subunits. Transmission-electron microscopy showed that STT3a becomes incorporated into OT-ribosome super-complexes formed in vivo, demonstrating that this expression/purification platform is suitable for analysis of large protein complexes. Pairwise in planta interaction analyses of individual OT subunits demonstrated that all subunits identified in animal OT complexes are conserved in Arabidopsis and physically interact with STT3a. Genetic analysis of newly established OT subunit mutants for OST1 and DEFENDER AGAINST APOTOTIC DEATH (DAD) family genes revealed that OST1 and DAD1/2 subunits are essential for the plant life cycle. However, mutations in these individual isoforms produced much milder growth/underglycosylation phenotypes than previously reported for mutations in DGL1, OST3/6 and STT3a.

Transcriptome-wide Analysis of Roles for tRNA Modifications in Translational Regulation.

Covalent nucleotide modifications in noncoding RNAs affect a plethora of biological processes, and new functions continue to be discovered even for well-known modifying enzymes. To systematically compare the functions of a large set of noncoding RNA modifications in gene regulation, we carried out ribosome profiling in budding yeast to characterize 57 nonessential genes involved in tRNA modification. Deletion mutants exhibited a range of translational phenotypes, with enzymes known to modify anticodons, or non-tRNA substrates such as rRNA, exhibiting the most dramatic translational perturbations. Our data build on prior reports documenting translational upregulation of the nutrient-responsive transcription factor Gcn4 in response to numerous tRNA perturbations, and identify many additional translationally regulated mRNAs throughout the yeast genome. Our data also uncover unexpected roles for tRNA-modifying enzymes in regulation of TY retroelements, and in rRNA 2'-O-methylation. This dataset should provide a rich resource for discovery of additional links between tRNA modifications and gene regulation.

Dissection of goadsporin biosynthesis by in vitro reconstitution leading to designer analogues expressed in vivo.

Goadsporin (GS) is a member of ribosomally synthesized and post-translationally modified peptides (RiPPs), containing an N-terminal acetyl moiety, six azoles and two dehydroalanines in the peptidic main chain. Although the enzymes involved in GS biosynthesis have been defined, the principle of how the respective enzymes control the specific modifications remains elusive. Here we report a one-pot synthesis of GS using the enzymes reconstituted in the 'flexible' in vitro translation system, referred to as the FIT-GS system. This system allows us to readily prepare not only the precursor peptide from its synthetic DNA template but also 52 mutants, enabling us to dissect the modification determinants of GodA for each enzyme. The in vitro knowledge has also led us to successfully produce designer GS analogues in vivo. The methodology demonstrated in this work is also applicable to other RiPP biosynthesis, allowing us to rapidly investigate the principle of modification events with great ease.

ZNF598 and RACK1 Regulate Mammalian Ribosome-Associated Quality Control Function by Mediating Regulatory 40S Ribosomal Ubiquitylation.

Ribosomes that experience terminal stalls during translation are resolved by ribosome-associated quality control (QC) pathways that oversee mRNA and nascent chain destruction and recycle ribosomal subunits. The proximal factors that sense stalled ribosomes and initiate mammalian ribosome-associated QC events remain undefined. We demonstrate that the ZNF598 ubiquitin ligase and the 40S ribosomal protein RACK1 help to resolve poly(A)-induced stalled ribosomes. They accomplish this by regulating distinct and overlapping regulatory 40S ribosomal ubiquitylation events. ZNF598 primarily mediates regulatory ubiquitylation of RPS10 and RPS20, whereas RACK1 regulates RPS2, RPS3, and RPS20 ubiquitylation. Gain or loss of ZNF598 function or mutations that block RPS10 or RPS20 ubiquitylation result in defective resolution of stalled ribosomes and subsequent readthrough of poly(A)-containing stall sequences. Together, our results indicate that ZNF598, RACK1, and 40S regulatory ubiquitylation plays a pivotal role in mammalian ribosome-associated QC pathways.

Nonconventional localizations of cytosolic aminoacyl-tRNA synthetases in yeast and human cells.

By definition, cytosolic aminoacyl-tRNA synthetases (aaRSs) should be restricted to the cytosol of eukaryotic cells where they supply translating ribosomes with their aminoacyl-tRNA substrates. However, it has been shown that other translationally-active compartments like mitochondria and plastids can simultaneously contain the cytosolic aaRS and its corresponding organellar ortholog suggesting that both forms do not share the same organellar function. In addition, a fair number of cytosolic aaRSs have also been found in the nucleus of cells from several species. Hence, these supposedly cytosolic-restricted enzymes have instead the potential to be multi-localized. As expected, in all examples that were studied so far, when the cytosolic aaRS is imported inside an organelle that already contains its bona fide corresponding organellar-restricted aaRSs, the cytosolic form was proven to exert a nonconventional and essential function. Some of these essential functions include regulating homeostasis and protecting against various stresses. It thus becomes critical to assess meticulously the subcellular localization of each of these cytosolic aaRSs to unravel their additional roles. With this objective in mind, we provide here a review on what is currently known about cytosolic aaRSs multi-compartmentalization and we describe all commonly used protocols and procedures for identifying the compartments in which cytosolic aaRSs relocalize in yeast and human cells.

Ribosomal Peptide Syntheses from Activated Substrates Reveal Rate Limitation by an Unexpected Step at the Peptidyl Site.

Protein synthesis (translation) is central to cellular function and antibiotic development. Interestingly, the key chemical step of translation, peptide bond formation, is among the slower enzymatic reactions. The reason for this remains controversial because of reliance on studies using highly modified, severely minimized, or unreactive substrate analogues. Here, we investigated this problem by fast kinetics using full-length aminoacyl-tRNA substrates with atomic substitutions that activated the ester electrophile. While trifluoro substitution of hydrogens in nonconserved positions of the peptidyl-site substrate dramatically increased the ester reactivity in solution assays, a large hastening of the combined rates of ribosomal accommodation and peptidyl transfer was observed only with a slowly reacting aminoacyl-site nucleophile, proline. With a fast-reacting A-site nucleophile, phenylalanine, effects did not correlate at all with electrophilicities. As effects were observed using the same, natural, aminoacyl-tRNA at the A site and all rates of accommodation/peptidyl transfer were pH dependent, we concluded that rate limitation was not by A-site accommodation but rather by peptidyl transfer and a hitherto unexpected step at the P site. This new slow step, which we term P-site accommodation, has implications for the activation or inhibition of ribosome function in vitro and in vivo.

The pathway to GTPase activation of elongation factor SelB on the ribosome.

In all domains of life, selenocysteine (Sec) is delivered to the ribosome by selenocysteine-specific tRNA (tRNASec) with the help of a specialized translation factor, SelB in bacteria. Sec-tRNASec recodes a UGA stop codon next to a downstream mRNA stem-loop. Here we present the structures of six intermediates on the pathway of UGA recoding in Escherichia coli by single-particle cryo-electron microscopy. The structures explain the specificity of Sec-tRNASec binding by SelB and show large-scale rearrangements of Sec-tRNASec. Upon initial binding of SelB-Sec-tRNASec to the ribosome and codon reading, the 30S subunit adopts an open conformation with Sec-tRNASec covering the sarcin-ricin loop (SRL) on the 50S subunit. Subsequent codon recognition results in a local closure of the decoding site, which moves Sec-tRNASec away from the SRL and triggers a global closure of the 30S subunit shoulder domain. As a consequence, SelB docks on the SRL, activating the GTPase of SelB. These results reveal how codon recognition triggers GTPase activation in translational GTPases.

Quantitative profiling of the in vivo enzymatic activity of ricin reveals disparate depurination of different pulmonary cell types.

The plant-derived toxins ricin and abrin, operate by site-specific depurination of ribosomes, which in turn leads to protein synthesis arrest. The clinical manifestation following pulmonary exposure to these toxins is that of a severe lung inflammation and respiratory insufficiency. Deciphering the pathways mediating between the catalytic activity and the developing lung inflammation, requires a quantitative appreciation of the catalytic activity of the toxins, in-vivo. In the present study, we monitored truncated cDNA molecules which are formed by reverse transcription when a depurinated 28S rRNA serves as template. We found that maximal depurination after intranasal exposure of mice to 2LD50 ricin was reached 48h, where nearly 40% of the ribosomes have been depurinated and that depurination can be halted by post-exposure administration of anti-ricin antibodies. We next demonstrated that the effect of ricin intoxication on different cell types populating the lungs differs greatly, and that outstandingly high levels of damage (80% depurination), were observed in particular for pulmonary epithelial cells. Finally, we found that the magnitude of depurination induced by the related plant-derived toxin abrin, was significantly lower in comparison to ricin, and can be attributed mostly to reduced depurination of pulmonary epithelial cells by abrin. This study provides for the first time vital information regarding the scope and timing of the catalytic performance of ricin and abrin in the lungs of intact animals.