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.

Sequestrase chaperones protect against oxidative stress-induced protein aggregation and [PSI+] prion formation.

Misfolded proteins are usually refolded to their functional conformations or degraded by quality control mechanisms. When misfolded proteins evade quality control, they can be sequestered to specific sites within cells to prevent the potential dysfunction and toxicity that arises from protein aggregation. Btn2 and Hsp42 are compartment-specific sequestrases that play key roles in the assembly of these deposition sites. Their exact intracellular functions and substrates are not well defined, particularly since heat stress sensitivity is not observed in deletion mutants. We show here that Btn2 and Hsp42 are required for tolerance to oxidative stress conditions induced by exposure to hydrogen peroxide. Btn2 and Hsp42 act to sequester oxidized proteins into defined PQC sites following ROS exposure and their absence leads to an accumulation of protein aggregates. The toxicity of protein aggregate accumulation causes oxidant sensitivity in btn2 hsp42 sequestrase mutants since overexpression of the Hsp104 disaggregase rescues oxidant tolerance. We have identified the Sup35 translation termination factor as an in vivo sequestrase substrate and show that Btn2 and Hsp42 act to suppress oxidant-induced formation of the yeast [PSI+] prion, which is the amyloid form of Sup35. [PSI+] prion formation in sequestrase mutants does not require IPOD (insoluble protein deposit) localization which is the site where amyloids are thought to undergo fragmentation and seeding to propagate their heritable prion form. Instead, both amorphous and amyloid Sup35 aggregates are increased in btn2 hsp42 mutants consistent with the idea that prion formation occurs at multiple intracellular sites during oxidative stress conditions in the absence of sequestrase activity. Taken together, our data identify protein sequestration as a key antioxidant defence mechanism that functions to mitigate the damaging consequences of protein oxidation-induced aggregation.

The ABI3-ERF1 module mediates ABA-auxin crosstalk to regulate lateral root emergence.

Abscisic acid (ABA) is involved in lateral root (LR) development, but how ABA signaling interacts with auxin signaling to regulate LR formation is not well understood. Here, we report that ABA-responsive ERF1 mediates the crosstalk between ABA and auxin signaling to regulate Arabidopsis LR emergence. ABI3 is a negative factor in LR emergence and transcriptionally activates ERF1 by binding to its promoter, and reciprocally, ERF1 activates ABI3, which forms a regulatory loop that enables rapid signal amplification. Notably, ABI3 physically interacts with ERF1, reducing the cis element-binding activities of both ERF1 and ABI3 and thus attenuating the expression of ERF1-/ABI3-regulated genes involved in LR emergence and ABA signaling, such as PIN1, AUX1, ARF7, and ABI5, which may provide a molecular rheostat to avoid overamplification of auxin and ABA signaling. Taken together, our findings identify the role of the ABI3-ERF1 module in mediating crosstalk between ABA and auxin signaling in LR emergence.

Discovery of new Lenalidomide derivatives as potent and selective GSPT1 degraders.

G1 to S phase transition 1 (GSPT1) is the requisite release factor for the translation termination. GSPT1 is identified as an oncogenic driver of several types of cancer and considered to be a promising cancer therapeutic target. Although two selective GSPT1 degraders were advanced into clinical trials, neither of them has been approved for clinical use. Here we developed a series of new selective GSPT1 degraders, among which the optimal compound 9q potently induced degradation of GSPT1 with a DC50 of 35 nM in U937 cells, and showed good selectivity in the global proteomic profiling study. Mechanism studies revealed that compound 9q induced GSPT1 degradation through the ubiquitin-proteasome system. Consistent with its potent GSPT1 degradation activity, compound 9q displayed good antiproliferative activities against U937 cells, MOLT-4 cells, and MV4-11 cells, with IC50 values of 0.019 µM, 0.006 µM, and 0.027 µM, respectively. Compound 9q also dose-dependently induced G0/G1 phase arrest and apoptosis in U937 cells.

Translation termination in human mitochondria - substrate specificity of mitochondrial release factors.

Mitochondria are the essential players in eukaryotic ATP production by oxidative phosphorylation, which relies on the maintenance and accurate expression of the mitochondrial genome. Even though the basic principles of translation are conserved due to the descendance from a bacterial ancestor, some deviations regarding translation factors as well as mRNA characteristics and the applied genetic code are present in human mitochondria. Together, these features are certain challenges during translation the mitochondrion has to handle. Here, we discuss the current knowledge regarding mitochondrial translation focusing on the termination process and the associated quality control mechanisms. We describe how mtRF1a resembles bacterial RF1 mechanistically and summarize in vitro and recent in vivo data leading to the conclusion of mtRF1a being the major mitochondrial release factor. On the other hand, we discuss the ongoing debate about the function of the second codon-dependent mitochondrial release factor mtRF1 regarding its role as a specialized termination factor. Finally, we link defects in mitochondrial translation termination to the activation of mitochondrial rescue mechanisms highlighting the importance of ribosome-associated quality control for sufficient respiratory function and therefore for human health.

Yeast Chaperone Hsp70-Ssb Modulates a Variety of Protein-Based Heritable Elements.

Prions are transmissible self-perpetuating protein isoforms associated with diseases and heritable traits. Yeast prions and non-transmissible protein aggregates (mnemons) are frequently based on cross-ß ordered fibrous aggregates (amyloids). The formation and propagation of yeast prions are controlled by chaperone machinery. Ribosome-associated chaperone Hsp70-Ssb is known (and confirmed here) to modulate formation and propagation of the prion form of the Sup35 protein [PSI+]. Our new data show that both formation and mitotic transmission of the stress-inducible prion form of the Lsb2 protein ([LSB+]) are also significantly increased in the absence of Ssb. Notably, heat stress leads to a massive accumulation of [LSB+] cells in the absence of Ssb, implicating Ssb as a major downregulator of the [LSB+]-dependent memory of stress. Moreover, the aggregated form of Gγ subunit Ste18, [STE+], behaving as a non-heritable mnemon in the wild-type strain, is generated more efficiently and becomes heritable in the absence of Ssb. Lack of Ssb also facilitates mitotic transmission, while lack of the Ssb cochaperone Hsp40-Zuo1 facilitates both spontaneous formation and mitotic transmission of the Ure2 prion, [URE3]. These results demonstrate that Ssb is a general modulator of cytosolic amyloid aggregation, whose effect is not restricted only to [PSI+].

Dynamics of release factor recycling during translation termination in bacteria.

In bacteria, release of newly synthesized proteins from ribosomes during translation termination is catalyzed by class-I release factors (RFs) RF1 or RF2, reading UAA and UAG or UAA and UGA codons, respectively. Class-I RFs are recycled from the post-termination ribosome by a class-II RF, the GTPase RF3, which accelerates ribosome intersubunit rotation and class-I RF dissociation. How conformational states of the ribosome are coupled to the binding and dissociation of the RFs remains unclear and the importance of ribosome-catalyzed guanine nucleotide exchange on RF3 for RF3 recycling in vivo has been disputed. Here, we profile these molecular events using a single-molecule fluorescence assay to clarify the timings of RF3 binding and ribosome intersubunit rotation that trigger class-I RF dissociation, GTP hydrolysis, and RF3 dissociation. These findings in conjunction with quantitative modeling of intracellular termination flows reveal rapid ribosome-dependent guanine nucleotide exchange to be crucial for RF3 action in vivo.

A Highly Substituted Ring-Fused 2-Pyridone Compound Targeting PrfA and the Efflux Regulator BrtA in Listeria monocytogenes.

Listeria monocytogenes is a facultative Gram-positive bacterium that causes listeriosis, a severe foodborne disease. We previously discovered that ring-fused 2-pyridone compounds can decrease virulence factor expression in Listeria by binding and inactivating the PrfA virulence activator. In this study, we tested PS900, a highly substituted 2-pyridone that was recently discovered to be bactericidal to other Gram-positive pathogenic bacteria, such as Staphylococcus aureus and Enterococcus faecalis. We show that PS900 can interact with PrfA and reduce the expression of virulence factors. Unlike previous ring-fused 2-pyridones shown to inactivate PrfA, PS900 had an additional antibacterial activity and was found to potentiate sensitivity toward cholic acid. Two PS900-tolerant mutants able to grow in the presence of PS900 carried mutations in the brtA gene, encoding the BrtA repressor. In wild-type (WT) bacteria, cholic acid binds and inactivates BrtA, thereby alleviating the expression of the multidrug transporter MdrT. Interestingly, we found that PS900 also binds to BrtA and that this interaction causes BrtA to dissociate from its binding site in front of the mdrT gene. In addition, we observed that PS900 potentiated the effect of different osmolytes. We suggest that the increased potency of cholic acid and osmolytes to kill bacteria in the presence of PS900 is due to the ability of the latter to inhibit general efflux, through a yet-unknown mechanism. Our data indicate that thiazolino 2-pyridones constitute an attractive scaffold when designing new types of antibacterial agents. IMPORTANCE Bacteria resistant to one or several antibiotics are a very large problem, threatening not only treatment of infections but also surgery and cancer treatments. Thus, new types of antibacterial drugs are desperately needed. In this work, we show that a new generation of substituted ring-fused 2-pyridones not only inhibit Listeria monocytogenes virulence gene expression, presumably by inactivating the PrfA virulence regulator, but also potentiate the bactericidal effects of cholic acid and different osmolytes. We identified a multidrug repressor as a second target of 2-pyridones. The repressor-2-pyridone interaction displaces the repressor from DNA, thus increasing the expression of a multidrug transporter. In addition, our data suggest that the new class of ring-fused 2-pyridones are efficient efflux inhibitors, possibly explaining why the simultaneous addition of 2-pyridones together with cholic acid or osmolytes is detrimental for the bacterium. This work proves conclusively that 2-pyridones constitute a promising scaffold to build on for future antibacterial drug design.

ATP modulates self-perpetuating conformational conversion generating structurally distinct yeast prion amyloids that limit autocatalytic amplification.

Prion-like self-perpetuating conformational conversion of proteins into amyloid aggregates is associated with both transmissible neurodegenerative diseases and non-Mendelian inheritance. The cellular energy currency ATP is known to indirectly regulate the formation, dissolution, or transmission of amyloid-like aggregates by providing energy to the molecular chaperones that maintain protein homeostasis. In this work, we demonstrate that ATP molecules, independent of any chaperones, modulate the formation and dissolution of amyloids from a yeast prion domain (NM domain of Saccharomyces cerevisiae Sup35) and restricts autocatalytic amplification by controlling the amount of fragmentable and seeding-competent aggregates. ATP, at (high) physiological concentrations in the presence of Mg2+, kinetically accelerates NM aggregation. Interestingly, ATP also promotes phase separation-mediated aggregation of a human protein harboring a yeast prion-like domain. We also show that ATP disaggregates preformed NM fibrils in a dose-independent manner. Our results indicate that ATP-mediated disaggregation, unlike the disaggregation by the disaggregase Hsp104, yields no oligomers that are considered one of the critical species for amyloid transmission. Furthermore, high concentrations of ATP delimited the number of seeds by giving rise to compact ATP-bound NM fibrils that exhibited nominal fragmentation by either free ATP or Hsp104 disaggregase to generate lower molecular weight amyloids. In addition, (low) pathologically relevant ATP concentrations restricted autocatalytic amplification by forming structurally distinct amyloids that are found seeding inefficient because of their reduced ß-content. Our results provide key mechanistic underpinnings of concentration-dependent chemical chaperoning by ATP against prion-like transmissions of amyloids.

Human mitochondria require mtRF1 for translation termination at non-canonical stop codons.

The mitochondrial translation machinery highly diverged from its bacterial counterpart. This includes deviation from the universal genetic code, with AGA and AGG codons lacking cognate tRNAs in human mitochondria. The locations of these codons at the end of COX1 and ND6 open reading frames, respectively, suggest they might function as stop codons. However, while the canonical stop codons UAA and UAG are known to be recognized by mtRF1a, the release mechanism at AGA and AGG codons remains a debated issue. Here, we show that upon the loss of another member of the mitochondrial release factor family, mtRF1, mitoribosomes accumulate specifically at AGA and AGG codons. Stalling of mitoribosomes alters COX1 transcript and protein levels, but not ND6 synthesis. In addition, using an in vitro reconstituted mitochondrial translation system, we demonstrate the specific peptide release activity of mtRF1 at the AGA and AGG codons. Together, our results reveal the role of mtRF1 in translation termination at non-canonical stop codons in mitochondria.

Short tRNA anticodon stem and mutant eRF1 allow stop codon reassignment.

Cognate tRNAs deliver specific amino acids to translating ribosomes according to the standard genetic code, and three codons with no cognate tRNAs serve as stop codons. Some protists have reassigned all stop codons as sense codons, neglecting this fundamental principle1-4. Here we analyse the in-frame stop codons in 7,259 predicted protein-coding genes of a previously undescribed trypanosomatid, Blastocrithidia nonstop. We reveal that in this species in-frame stop codons are underrepresented in genes expressed at high levels and that UAA serves as the only termination codon. Whereas new tRNAsGlu fully cognate to UAG and UAA evolved to reassign these stop codons, the UGA reassignment followed a different path through shortening the anticodon stem of tRNATrpCCA from five to four base pairs (bp). The canonical 5-bp tRNATrp recognizes UGG as dictated by the genetic code, whereas its shortened 4-bp variant incorporates tryptophan also into in-frame UGA. Mimicking this evolutionary twist by engineering both variants from B. nonstop, Trypanosoma brucei and Saccharomyces cerevisiae and expressing them in the last two species, we recorded a significantly higher readthrough for all 4-bp variants. Furthermore, a gene encoding B. nonstop release factor 1 acquired a mutation that specifically restricts UGA recognition, robustly potentiating the UGA reassignment. Virtually the same strategy has been adopted by the ciliate Condylostoma magnum. Hence, we describe a previously unknown, universal mechanism that has been exploited in unrelated eukaryotes with reassigned stop codons.

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.

Hsp40/JDP Requirements for the Propagation of Synthetic Yeast Prions.

Yeast prions are protein-based transmissible elements, most of which are amyloids. The chaperone protein network in yeast is inexorably linked to the spreading of prions during cell division by fragmentation of amyloid prion aggregates. Specifically, the core "prion fragmentation machinery" includes the proteins Hsp104, Hsp70 and the Hsp40/J-domain protein (JDP) Sis1. Numerous novel amyloid-forming proteins have been created and examined in the yeast system and occasionally these amyloids are also capable of continuous Hsp104-dependent propagation in cell populations, forming synthetic prions. However, additional chaperone requirements, if any, have not been determined. Here, we report the first instances of a JDP-Hsp70 system requirement for the propagation of synthetic prions. We utilized constructs from a system of engineered prions with prion-forming domains (PrDs) consisting of a polyQ stretch interrupted by a single heterologous amino acid interspersed every fifth residue. These "polyQX" PrDs are fused to the MC domains of Sup35, creating chimeric proteins of which a subset forms synthetic prions in yeast. For four of these prions, we show that SIS1 repression causes prion loss in a manner consistent with Sis1's known role in prion fragmentation. PolyQX prions were sensitive to Sis1 expression levels to differing degrees, congruent with the variability observed among native prions. Our results expand the scope known Sis1 functionality, demonstrating that Sis1 acts on amyloids broadly, rather than through specific protein-protein interactions with individual yeast prion-forming proteins.

Rate-limiting hydrolysis in ribosomal release reactions revealed by ester activation.

Translation terminates by releasing the polypeptide chain in one of two chemical reactions catalyzed by the ribosome. Release is also a target for engineering, as readthrough of a stop codon enables incorporation of unnatural amino acids and treatment of genetic diseases. Hydrolysis of the ester bond of peptidyl-tRNA requires conformational changes of both a class I release factor (RF) protein and the peptidyl transferase center of a large subunit rRNA. The rate-limiting step was proposed to be hydrolysis at physiological pH and an RF conformational change at higher pH, but evidence was indirect. Here, we tested this by activating the ester electrophile at the Escherichia coli ribosomal P site using a trifluorine-substituted amino acid. Quench-flow kinetics revealed that RF1-catalyzed release could be accelerated, but only at pH 6.2-7.7 and not higher pH. This provided direct evidence for rate-limiting hydrolysis at physiological or lower pH and a different rate limitation at higher pH. Additionally, we optimized RF-free release catalyzed by unacylated tRNA or the CCA trinucleotide (in 30% acetone). We determined that these two model release reactions, although very slow, were surprisingly accelerated by the trifluorine analog but to a different extent from each other and from RF-catalyzed release. Hence, hydrolysis was rate limiting in all three reactions. Furthermore, in 20% ethanol, we found that there was significant competition between fMet-ethyl ester formation and release in all three release reactions. We thus favor proposed mechanisms for translation termination that do not require a fully-negatively-charged OH- nucleophile.

Human mtRF1 terminates COX1 translation and its ablation induces mitochondrial ribosome-associated quality control.

Translation termination requires release factors that read a STOP codon in the decoding center and subsequently facilitate the hydrolysis of the nascent peptide chain from the peptidyl tRNA within the ribosome. In human mitochondria eleven open reading frames terminate in the standard UAA or UAG STOP codon, which can be recognized by mtRF1a, the proposed major mitochondrial release factor. However, two transcripts encoding for COX1 and ND6 terminate in the non-conventional AGA or AGG codon, respectively. How translation termination is achieved in these two cases is not known. We address this long-standing open question by showing that the non-canonical release factor mtRF1 is a specialized release factor that triggers COX1 translation termination, while mtRF1a terminates the majority of other mitochondrial translation events including the non-canonical ND6. Loss of mtRF1 leads to isolated COX deficiency and activates the mitochondrial ribosome-associated quality control accompanied by the degradation of COX1 mRNA to prevent an overload of the ribosome rescue system. Taken together, these results establish the role of mtRF1 in mitochondrial translation, which had been a mystery for decades, and lead to a comprehensive picture of translation termination in human mitochondria.

Role of FruR transcriptional regulator in virulence of Listeria monocytogenes and identification of its regulon.

Listeria monocytogenes is a ubiquitous opportunistic foodborne pathogen capable of survival in various adverse environmental conditions. Pathogenesis of L. monocytogenes is tightly controlled by a complex regulatory network of transcriptional regulators that are necessary for survival and adaptations to harsh environmental conditions both inside and outside host cells. Among these regulatory pathways are members of the DeoR-family transcriptional regulators that are known to play a regulatory role in sugar metabolism. In this study, we deciphered the role of FruR, a DeoR family protein, which is a fructose operon transcriptional repressor protein, in L. monocytogenes pathogenesis and growth. Following intravenous (IV) inoculation in mice, a mutant strain with deletion of fruR exhibited a significant reduction in bacterial burden in liver and spleen tissues compared to the parent strain. Further, the ΔfruR strain had a defect in cell-to-cell spread in L2 fibroblast monolayers. Constitutive activation of PrfA, a pleiotropic activator of L. monocytogenes virulence factors, did not restore virulence to the ΔfruR strain, suggesting that the attenuation was not a result of impaired PrfA activation. Transcriptome analysis revealed that FruR functions as a positive regulator for genes encoding enzymes involved in the pentose phosphate pathway (PPP) and as a repressor for genes encoding enzymes in the glycolysis pathway. These results suggested that FruR may function to facilitate NADPH regeneration, which is necessary for full protection from oxidative stress. Interestingly, deletion of fruR increased sensitivity of L. monocytogenes to H2O2, confirming a role for FruR in survival of L. monocytogenes during oxidative stress. Using anti-mouse neutrophil/monocyte monoclonal antibody RB6-8C5 (RB6) in an in vivo infection model, we found that FruR has a specific function in protecting L. monocytogenes from neutrophil/monocyte-mediated killing. Overall, this work clarifies the role of FruR in controlling L. monocytogenes carbon flow between glycolysis and PPP for NADPH homeostasis, which provides a new mechanism allowing metabolic adaptation of L. monocytogenes to oxidative stress.

The [PSI+] prion modulates cytochrome c oxidase deficiency caused by deletion of COX12.

Cytochrome c oxidase (CcO) is a pivotal enzyme of the mitochondrial respiratory chain, which sustains bioenergetics of eukaryotic cells. Cox12, a peripheral subunit of CcO oxidase, is required for full activity of the enzyme, but its exact function is unknown. Here experimental evolution of a Saccharomyces cerevisiae Δcox12 strain for ∼300 generations allowed to restore the activity of CcO oxidase. In one population, the enhanced bioenergetics was caused by a A375V mutation in the cytosolic AAA+ disaggregase Hsp104. Deletion or overexpression of HSP104 also increased respiration of the Δcox12 ancestor strain. This beneficial effect of Hsp104 was related to the loss of the [PSI+] prion, which forms cytosolic amyloid aggregates of the Sup35 protein. Overall, our data demonstrate that cytosolic aggregation of a prion impairs the mitochondrial metabolism of cells defective for Cox12. These findings identify a new functional connection between cytosolic proteostasis and biogenesis of the mitochondrial respiratory chain.

Proximity interactome analysis of Lassa polymerase reveals eRF3a/GSPT1 as a druggable target for host-directed antivirals.

Completion of the Lassa virus (LASV) life cycle critically depends on the activities of the virally encoded, RNA-dependent RNA polymerase in replication and transcription of the viral RNA genome in the cytoplasm of infected cells. The contribution of cellular proteins to these processes remains unclear. Here, we applied proximity proteomics to define the interactome of LASV polymerase in cells under conditions that recreate LASV RNA synthesis. We engineered a LASV polymerase-biotin ligase (TurboID) fusion protein that retained polymerase activity and successfully biotinylated the proximal proteome, which allowed the identification of 42 high-confidence LASV polymerase interactors. We subsequently performed a small interfering RNA (siRNA) screen to identify those interactors that have functional roles in authentic LASV infection. As proof of principle, we characterized eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1), which we found to be a proviral factor that physically associates with LASV polymerase. Targeted degradation of GSPT1 by a small-molecule drug candidate, CC-90009, resulted in strong inhibition of LASV infection in cultured cells. Our work demonstrates the feasibility of using proximity proteomics to illuminate and characterize yet-to-be-defined host-pathogen interactome, which can reveal new biology and uncover novel targets for the development of antivirals against highly pathogenic RNA viruses.

Small-molecule eRF3a degraders rescue CFTR nonsense mutations by promoting premature termination codon readthrough.

The vast majority of people with cystic fibrosis (CF) are now eligible for CF transmembrane regulator (CFTR) modulator therapy. The remaining individuals with CF harbor premature termination codons (PTCs) or rare CFTR variants with limited treatment options. Although the clinical modulator response can be reliably predicted using primary airway epithelial cells, primary cells carrying rare CFTR variants are scarce. To overcome this obstacle, cell lines can be created by overexpression of mouse Bmi-1 and human TERT (hTERT). Using this approach, we developed 2 non-CF and 6 CF airway epithelial cell lines, 3 of which were homozygous for the W1282X PTC variant. The Bmi-1/hTERT cell lines recapitulated primary cell morphology and ion transport function. The 2 F508del-CFTR cell lines responded robustly to CFTR modulators, which was mirrored in the parent primary cells and in the cell donors' clinical response. Cereblon E3 ligase modulators targeting eukaryotic release factor 3a (eRF3a) rescued W1282X-CFTR function to approximately 20% of WT levels and, when paired with G418, rescued G542X-CFTR function to approximately 50% of WT levels. Intriguingly, eRF3a degraders also diminished epithelial sodium channel (ENaC) function. These studies demonstrate that Bmi-1/hTERT cell lines faithfully mirrored primary cell responses to CFTR modulators and illustrate a therapeutic approach to rescue CFTR nonsense mutations.

Implications of the Actin Cytoskeleton on the Multi-Step Process of [PSI+] Prion Formation.

Yeast prions are self-perpetuating misfolded proteins that are infectious. In yeast, [PSI+] is the prion form of the Sup35 protein. While the study of [PSI+] has revealed important cellular mechanisms that contribute to prion propagation, the underlying cellular factors that influence prion formation are not well understood. Prion formation has been described as a multi-step process involving both the initial nucleation and growth of aggregates, followed by the subsequent transmission of prion particles to daughter cells. Prior evidence suggests that actin plays a role in this multi-step process, but actin's precise role is unclear. Here, we investigate how actin influences the cell's ability to manage newly formed visible aggregates and how actin influences the transmission of newly formed aggregates to future generations. At early steps, using 3D time-lapse microscopy, several actin mutants, and Markov modeling, we find that the movement of newly formed aggregates is random and actin independent. At later steps, our prion induction studies provide evidence that the transmission of newly formed prion particles to daughter cells is limited by the actin cytoskeletal network. We suspect that this limitation is because actin is used to possibly retain prion particles in the mother cell.