A new family of bacterial ribosome hibernation factors.

To conserve energy during starvation and stress, many organisms use hibernation factor proteins to inhibit protein synthesis and protect their ribosomes from damage1,2. In bacteria, two families of hibernation factors have been described, but the low conservation of these proteins and the huge diversity of species, habitats and environmental stressors have confounded their discovery3-6. Here, by combining cryogenic electron microscopy, genetics and biochemistry, we identify Balon, a new hibernation factor in the cold-adapted bacterium Psychrobacter urativorans. We show that Balon is a distant homologue of the archaeo-eukaryotic translation factor aeRF1 and is found in 20% of representative bacteria. During cold shock or stationary phase, Balon occupies the ribosomal A site in both vacant and actively translating ribosomes in complex with EF-Tu, highlighting an unexpected role for EF-Tu in the cellular stress response. Unlike typical A-site substrates, Balon binds to ribosomes in an mRNA-independent manner, initiating a new mode of ribosome hibernation that can commence while ribosomes are still engaged in protein synthesis. Our work suggests that Balon-EF-Tu-regulated ribosome hibernation is a ubiquitous bacterial stress-response mechanism, and we demonstrate that putative Balon homologues in Mycobacteria bind to ribosomes in a similar fashion. This finding calls for a revision of the current model of ribosome hibernation inferred from common model organisms and holds numerous implications for how we understand and study ribosome hibernation.

Molecular basis of translation termination at noncanonical stop codons in human mitochondria.

The genetic code that specifies the identity of amino acids incorporated into proteins during protein synthesis is almost universally conserved. Mitochondrial genomes feature deviations from the standard genetic code, including the reassignment of two arginine codons to stop codons. The protein required for translation termination at these noncanonical stop codons to release the newly synthesized polypeptides is not currently known. In this study, we used gene editing and ribosomal profiling in combination with cryo-electron microscopy to establish that mitochondrial release factor 1 (mtRF1) detects noncanonical stop codons in human mitochondria by a previously unknown mechanism of codon recognition. We discovered that binding of mtRF1 to the decoding center of the ribosome stabilizes a highly unusual conformation in the messenger RNA in which the ribosomal RNA participates in specific recognition of the noncanonical stop codons.

Hsp70 Binding to the N-terminal Domain of Hsp104 Regulates [PSI+] Curing by Hsp104 Overexpression.

Hsp104 propagates the yeast prion [PSI+], the infectious form of Sup35, by severing the prion seeds, but when Hsp104 is overexpressed, it cures [PSI+] in a process that is not yet understood but may be caused by trimming, which removes monomers from the ends of the amyloid fibers. This curing was shown to depend on both the N-terminal domain of Hsp104 and the expression level of various members of the Hsp70 family, which raises the question as to whether these effects of Hsp70 are due to it binding to the Hsp70 binding site that was identified in the N-terminal domain of Hsp104, a site not involved in prion propagation. Investigating this question, we now find, first, that mutating this site prevents both the curing of [PSI+] by Hsp104 overexpression and the trimming activity of Hsp104. Second, we find that depending on the specific member of the Hsp70 family binding to the N-terminal domain of Hsp104, both trimming and the curing caused by Hsp104 overexpression are either increased or decreased in parallel. Therefore, the binding of Hsp70 to the N-terminal domain of Hsp104 regulates both the rate of [PSI+] trimming by Hsp104 and the rate of [PSI+] curing by Hsp104 overexpression.

The Mutability of Yeast Prions.

Prions replicate by a self-templating mechanism. Infidelity in the process can lead to the emergence of new infectious structures, referred to as variants or strains. The question of whether prions are prone to mis-templating is not completely answered. Our previous experiments with 23 variants of the yeast [PSI+] prion do not support broad mutability. However, it became clear recently that the heat shock protein Hsp104 can restrict [PSI+] strain variation. This raises the possibility that many transmutable variants of the prion may have been mistaken as faithful-propagating simply because the mutant structure was too sturdy or too frail to take root in the wild-type cell. Here, I alter the strength of Hsp104 in yeast, overexpressing wild-type Hsp104 or expressing the hypo-active Hsp104T160M mutant, and check if the new environments enable the variants to mutate. Two variants hitherto thought of as faithful-propagating are discovered to generate different structures, which are stabilized with the hypo-active chaperone. In contrast, most transmutable variants discovered in cells overexpressing Hsp104 have been correctly identified as such previously in wild-type cells without the overexpression. The majority of transmutable variants only mis-template the structure of VH, VK, or VL, which are the most frequently observed variants and do not spontaneously mutate. There are four additional variants that never give rise to different structures in all cell conditions tested. Therefore, quite a few [PSI+] variants are faithful-propagating, and even the transmutable ones do not freely evolve but can only change to limited structural types.

Degradation of GSPT1 causes TP53-independent cell death in leukemia while sparing normal hematopoietic stem cells.

Targeted protein degradation is a rapidly advancing and expanding therapeutic approach. Drugs that degrade GSPT1 via the CRL4CRBN ubiquitin ligase are a new class of cancer therapy in active clinical development with evidence of activity against acute myeloid leukemia in early-phase trials. However, other than activation of the integrated stress response, the downstream effects of GSPT1 degradation leading to cell death are largely undefined, and no murine models are available to study these agents. We identified the domains of GSPT1 essential for cell survival and show that GSPT1 degradation leads to impaired translation termination, activation of the integrated stress response pathway, and TP53-independent cell death. CRISPR/Cas9 screens implicated decreased translation initiation as protective following GSPT1 degradation, suggesting that cells with higher levels of translation are more susceptible to the effects of GSPT1 degradation. We defined 2 Crbn amino acids that prevent Gspt1 degradation in mice, generated a knockin mouse with alteration of these residues, and demonstrated the efficacy of GSPT1-degrading drugs in vivo with relative sparing of numbers and function of long-term hematopoietic stem cells. Our results provide a mechanistic basis for the use of GSPT1 degraders for the treatment of cancer, including TP53-mutant acute myeloid leukemia.

Nsp1 of SARS-CoV-2 stimulates host translation termination.

Nsp1 of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of the Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1, eRF1 and ABCE1. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates peptide release and formation of termination complexes. Detailed analysis of Nsp1 activity during translation termination stages reveals that Nsp1 facilitates stop codon recognition. We demonstrate that Nsp1 stimulation targets eRF1 and does not affect eRF3. Moreover, Nsp1 increases amount of the termination complexes at all three stop codons. The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that the biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and remove them from the pool of the active ribosomes.

Structural basis of l-tryptophan-dependent inhibition of release factor 2 by the TnaC arrest peptide.

In Escherichia coli, elevated levels of free l-tryptophan (l-Trp) promote translational arrest of the TnaC peptide by inhibiting its termination. However, the mechanism by which translation-termination by the UGA-specific decoding release factor 2 (RF2) is inhibited at the UGA stop codon of stalled TnaC-ribosome-nascent chain complexes has so far been ambiguous. This study presents cryo-EM structures for ribosomes stalled by TnaC in the absence and presence of RF2 at average resolutions of 2.9 and 3.5 Å, respectively. Stalled TnaC assumes a distinct conformation composed of two small α-helices that act together with residues in the peptide exit tunnel (PET) to coordinate a single L-Trp molecule. In addition, while the peptidyl-transferase center (PTC) is locked in a conformation that allows RF2 to adopt its canonical position in the ribosome, it prevents the conserved and catalytically essential GGQ motif of RF2 from adopting its active conformation in the PTC. This explains how translation of the TnaC peptide effectively allows the ribosome to function as a L-Trp-specific small-molecule sensor that regulates the tnaCAB operon.

GGQ methylation enhances both speed and accuracy of stop codon recognition by bacterial class-I release factors.

Accurate translation termination in bacteria requires correct recognition of the stop codons by the class-I release factors (RFs) RF1 and RF2, which release the nascent peptide from the peptidyl tRNA after undergoing a "compact to open" conformational transition. These RFs possess a conserved Gly-Gly-Gln (GGQ) peptide release motif, of which the Q residue is posttranslationally methylated. GGQ-methylated RFs have been shown to be faster in peptide release than the unmethylated ones, but it was unknown whether this modification had additional roles. Using a fluorescence-based real-time in vitro translation termination assay in a stopped-flow instrument, we demonstrate that methylated RF1 and RF2 are two- to four-fold more accurate in the cognate stop codon recognition than their unmethylated variants. Using pH titration, we show that the lack of GGQ methylation facilitates the "compact to open" transition, which results in compromised accuracy of the unmethylated RFs. Furthermore, thermal melting studies using circular dichroism and SYPRO-orange fluorescence demonstrate that GGQ methylation increases overall stability of the RF proteins. This increased stability, we suspect, is the basis for the more controlled conformational change of the methylated RFs upon codon recognition, which enhances both their speed and accuracy. This GGQ methylation-based modulation of the accuracy of RFs can be a tool for regulating translational termination in vivo.

CC-90009: A Cereblon E3 Ligase Modulating Drug That Promotes Selective Degradation of GSPT1 for the Treatment of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is marked by significant unmet clinical need due to both poor survival and high relapse rates where long-term disease control for most patients with relapsed or refractory AML remain dismal. Inspired to bring novel therapeutic options to these patients, we envisioned protein degradation as a potential therapeutic approach for the treatment of AML. Following this course, we discovered and pioneered a novel mechanism of action which culminated in the discovery of CC-90009. CC-90009 represents a novel protein degrader and the first cereblon E3 ligase modulating drug to enter clinical development that specifically targets GSPT1 (G1 to S phase transition 1) for proteasomal degradation. This manuscript briefly summarizes the mechanism of action, scientific rationale, medicinal chemistry, pharmacokinetic properties, and efficacy data for CC-90009, which is currently in phase 1 clinical development.

Michler's hydrol blue elucidates structural differences in prion strains.

Yeast prions provide self-templating protein-based mechanisms of inheritance whose conformational changes lead to the acquisition of diverse new phenotypes. The best studied of these is the prion domain (NM) of Sup35, which forms an amyloid that can adopt several distinct conformations (strains) that confer distinct phenotypes when introduced into cells that do not carry the prion. Classic dyes, such as thioflavin T and Congo red, exhibit large increases in fluorescence when bound to amyloids, but these dyes are not sensitive to local structural differences that distinguish amyloid strains. Here we describe the use of Michler's hydrol blue (MHB) to investigate fibrils formed by the weak and strong prion fibrils of Sup35NM and find that MHB differentiates between these two polymorphs. Quantum mechanical time-dependent density functional theory (TDDFT) calculations indicate that the fluorescence properties of amyloid-bound MHB can be correlated to the change of binding site polarity and that a tyrosine to phenylalanine substitution at a binding site could be detected. Through the use of site-specific mutants, we demonstrate that MHB is a site-specific environmentally sensitive probe that can provide structural details about amyloid fibrils and their polymorphs.

Elongational stalling activates mitoribosome-associated quality control.

The human mitochondrial ribosome (mitoribosome) and associated proteins regulate the synthesis of 13 essential subunits of the oxidative phosphorylation complexes. We report the discovery of a mitoribosome-associated quality control pathway that responds to interruptions during elongation, and we present structures at 3.1- to 3.3-angstrom resolution of mitoribosomal large subunits trapped during ribosome rescue. Release factor homolog C12orf65 (mtRF-R) and RNA binding protein C6orf203 (MTRES1) eject the nascent chain and peptidyl transfer RNA (tRNA), respectively, from stalled ribosomes. Recruitment of mitoribosome biogenesis factors to these quality control intermediates suggests additional roles for these factors during mitoribosome rescue. We also report related cryo-electron microscopy structures (3.7 to 4.4 angstrom resolution) of elongating mitoribosomes bound to tRNAs, nascent polypeptides, the guanosine triphosphatase elongation factors mtEF-Tu and mtEF-G1, and the Oxa1L translocase.

A Tale of Two Transitions: The Unfolding Mechanism of the prfA RNA Thermosensor.

RNA thermosensors (RNATs), found in the 5' untranslated region (UTR) of some bacterial messenger RNAs (mRNAs), control the translation of the downstream gene in a temperature-dependent manner. In Listeria monocytogenes, the expression of a key transcription factor, PrfA, is mediated by an RNAT in its 5' UTR. PrfA functions as a master regulator of virulence in L. monocytogenes, controlling the expression of many virulence factors. The temperature-regulated expression of PrfA by its RNAT element serves as a signal of successful host invasion for the bacteria. Structurally, the prfA RNAT bears little resemblance to known families of RNATs, and prior studies demonstrated that the prfA RNAT is highly responsive over a narrow temperature range. Herein, we have undertaken a comprehensive mutational and thermodynamic analysis to ascertain the molecular determinants of temperature sensitivity. We provide evidence to support the idea that the prfA RNAT unfolding is different from that of cssA, a well-characterized RNAT, suggesting that these RNATs function via distinct mechanisms. Our data show that the unfolding of the prfA RNAT occurs in two distinct events and that the internal loops play an important role in mediating the cooperativity of RNAT unfolding. We further demonstrated that regions distal to the ribosome binding site (RBS) not only contribute to RNAT structural stability but also impact translation of the downstream message. Our collective results provide insight connecting the thermal stability of the prfA RNAT structure, unfolding energetics, and translational control.

Selective Degradation of GSPT1 by Cereblon Modulators Identified via a Focused Combinatorial Library.

Cereblon (CRBN) is an E3 ligase adapter protein that can be reprogrammed by imide-class compounds such as thalidomide, lenalidomide, and pomalidomide to induce the degradation of neo-substrate proteins. In order to identify additional small molecule CRBN modulators, we implemented a focused combinatorial library approach where we fused an imide-based CRBN-binding pharmacophore to a heterocyclic scaffold, which could be further elaborated. We screened the library for CRBN-dependent antiproliferative activity in the multiple myeloma cell line MM1.S and identified five hit compounds. Quantitative chemical proteomics of hit compounds revealed that they induced selective degradation of GSPT1, a translation termination factor that is currently being explored as a therapeutic target for the treatment of acute myeloid leukemia. Molecular docking studies with CRBN and GSPT1 followed by analogue synthesis identified a possible hydrogen bond interaction with the central pyrimidine ring as a molecular determinant of hit compounds' selectivity. This study demonstrates that a focused combinatorial library design, phenotypic screening, and chemical proteomics can provide a suitable workflow to efficiently identify novel CRBN modulators.

The Yarrowia lipolytica orthologs of Sup35p assemble into thioflavin T-negative amyloid fibrils.

The translation terminator Sup35p assembles into self-replicating fibrillar aggregates that are responsible for the [PSI+] prion state. The Q/N-rich N-terminal domain together with the highly charged middle-domain (NM domain) drive the assembly of Sup35p into amyloid fibrils in vitro. NM domains are highly divergent among yeasts. The ability to convert to a prion form is however conserved among Sup35 orthologs. In particular, the Yarrowia lipolytica Sup35p stands out with an exceptionally high prion conversion rate. In the present work, we show that different Yarrowia lipolytica strains contain one of two Sup35p orthologs that differ by the number of repeats within their NM domain. The Y. lipolytica Sup35 proteins are able to assemble into amyloid fibrils. Contrary to S. cerevisiae Sup35p, fibrils made of full-length or NM domains of Y. lipolytica Sup35 proteins did not bind Thioflavin-T, a well-known marker of amyloid aggregates.

Nucleation seed size determines amyloid clearance and establishes a barrier to prion appearance in yeast.

Amyloid appearance is a rare event that is promoted in the presence of other aggregated proteins. These aggregates were thought to act by templating the formation of an assembly-competent nucleation seed, but we find an unanticipated role for them in enhancing the persistence of amyloid after it arises. Specifically, Saccharomyces cerevisiae Rnq1 amyloid reduces chaperone-mediated disassembly of Sup35 amyloid, promoting its persistence in yeast. Mathematical modeling and corresponding in vivo experiments link amyloid persistence to the conformationally defined size of the Sup35 nucleation seed and suggest that amyloid is actively cleared by disassembly below this threshold to suppress appearance of the [PSI+] prion in vivo. Remarkably, this framework resolves multiple known inconsistencies in the appearance and curing of yeast prions. Thus, our observations establish the size of the nucleation seed as a previously unappreciated characteristic of prion variants that is key to understanding transitions between prion states.

Short disordered protein segment regulates cross-species transmission of a yeast prion.

Soluble prion proteins contingently encounter foreign prion aggregates, leading to cross-species prion transmission. However, how its efficiency is regulated by structural fluctuation of the host soluble prion protein remains unsolved. In the present study, through the use of two distantly related yeast prion Sup35 proteins, we found that a specific conformation of a short disordered segment governs interspecies prion transmissibility. Using a multidisciplinary approach including high-resolution NMR and molecular dynamics simulation, we identified critical residues within this segment that allow interspecies prion transmission in vitro and in vivo, by locally altering dynamics and conformation of soluble prion proteins. Remarkably, subtle conformational differences caused by a methylene group between asparagine and glutamine sufficed to change the short segment structure and substantially modulate the cross-seeding activity. Thus, our findings uncover how conformational dynamics of the short segment in the host prion protein impacts cross-species prion transmission. More broadly, our study provides mechanistic insights into cross-seeding between heterologous proteins.

Dynamics of oligomer and amyloid fibril formation by yeast prion Sup35 observed by high-speed atomic force microscopy.

The yeast prion protein Sup35, which contains intrinsically disordered regions, forms amyloid fibrils responsible for a prion phenotype [PSI+]. Using high-speed atomic force microscopy (HS-AFM), we directly visualized the prion determinant domain (Sup35NM) and the formation of its oligomers and fibrils at subsecond and submolecular resolutions. Monomers with freely moving tail-like regions initially appeared in the images, and subsequently oligomers with distinct sizes of ∼1.7 and 3 to 4 nm progressively accumulated. Nevertheless, these oligomers did not form fibrils, even after an incubation for 2 h in the presence of monomers. Fibrils appeared after much longer monomer incubation. The fibril elongation occurred smoothly without discrete steps, suggesting gradual conversions of the incorporated monomers into cross-ß structures. The individual oligomers were separated from each other and also from the fibrils by respective, identical lengths on the mica surface, probably due to repulsion caused by the freely moving disordered regions. Based on these HS-AFM observations, we propose that the freely moving tails of the monomers are incorporated into the fibril ends, and then the structural conversions to cross-ß structures gradually occur.

Ornithine capture by a translating ribosome controls bacterial polyamine synthesis.

Polyamines are essential metabolites that play an important role in cell growth, stress adaptation and microbial virulence1-3. To survive and multiply within a human host, pathogenic bacteria adjust the expression and activity of polyamine biosynthetic enzymes in response to different environmental stresses and metabolic cues2. Here, we show that ornithine capture by the ribosome and the nascent peptide SpeFL controls polyamine synthesis in γ-proteobacteria by inducing the expression of the ornithine decarboxylase SpeF4, via a mechanism involving ribosome stalling and transcription antitermination. In addition, we present the cryogenic electron microscopy structure of an Escherichia coli ribosome stalled during translation of speFL in the presence of ornithine. The structure shows how the ribosome and the SpeFL sensor domain form a highly selective binding pocket that accommodates a single ornithine molecule but excludes near-cognate ligands. Ornithine pre-associates with the ribosome and is then held in place by the sensor domain, leading to the compaction of the SpeFL effector domain and blocking the action of release factor 1. Thus, our study not only reveals basic strategies by which nascent peptides assist the ribosome in detecting a specific metabolite, but also provides a framework for assessing how ornithine promotes virulence in several human pathogens.

Yeast Tripartite Biosensors Sensitive to Protein Stability and Aggregation Propensity.

In contrast to the myriad approaches available to study protein misfolding and aggregation in vitro, relatively few tools are available for the study of these processes in the cellular context. This is in part due to the complexity of the cellular environment which, for instance, interferes with many spectroscopic approaches. Here, we describe a tripartite fusion approach that can be used to assess in vivo protein stability and solubility in the cytosol of Saccharomyces cerevisiae. Our biosensors contain tripartite fusions in which a protein of interest is inserted into antibiotic resistance markers. These fusions act to directly link the aggregation susceptibility and stability of the inserted protein to antibiotic resistance. We demonstrate a linear relationship between the thermodynamic stabilities of variants of the model folding protein immunity protein 7 (Im7) fused into the resistance markers and their antibiotic resistance readouts. We also use this system to investigate the in vivo properties of the yeast prion proteins Sup35 and Rnq1 and proteins whose aggregation is associated with some of the most prevalent neurodegenerative misfolding disorders, including peptide amyloid beta 1-42 (Aß42), which is involved in Alzheimer's disease, and protein α-synuclein, which is linked to Parkinson's disease.

Targeted protein degradation: current and future challenges.

Traditional approaches in the development of small-molecule drugs typically aim to inhibit the biochemical activity of functional protein domains. In contrast, targeted protein degradation aims to reduce overall levels of disease-relevant proteins. Mechanistically, this can be achieved via chemical ligands that induce molecular proximity between an E3 ubiquitin ligase and a protein of interest, leading to ubiquitination and degradation of the protein of interest. This paradigm-shifting pharmacology promises to address several limitations inherent to conventional inhibitor design. Most notably, targeted protein degradation has the potential not only to expand the druggable proteome beyond the reach of traditional competitive inhibitors but also to develop therapeutic strategies of unmatched selectivity. This review briefly summarizes key challenges that remain to be addressed to deliver on these promises and to realize the full therapeutic potential of pharmacologic modulation of protein degradation pathways.