The synthetic aminoglycoside ELX-02 induces readthrough of G550X-CFTR producing superfunctional protein that can be further enhanced by CFTR modulators.

Ten percent of cystic fibrosis (CF) patients carry a premature termination codon (PTC); no mutation-specific therapies exist for these individuals. ELX-02, a synthetic aminoglycoside, suppresses translation termination at PTCs (i.e., readthrough) by promoting the insertion of an amino acid at the PTC and restoring expression of full-length CFTR protein. The identity of amino acids inserted at PTCs affects the processing and function of the resulting full-length CFTR protein. We examined readthrough of the rare G550X-CFTR nonsense mutation due to its unique properties. We found that forskolin-induced swelling in G550X patient-derived intestinal organoids (PDOs) was significantly higher than in G542X PDOs (both UGA PTCs) with ELX-02 treatment, indicating greater CFTR function from the G550X allele. Using mass spectrometry, we identified tryptophan as the sole amino acid inserted in the G550X position during ELX-02- or G418-mediated readthrough, which differs from the three amino acids (cysteine, arginine, and tryptophan) inserted in the G542X position after treatment with G418. Compared with wild-type CFTR, Fischer rat thyroid (FRT) cells expressing the G550W-CFTR variant protein exhibited significantly increased forskolin-activated Cl- conductance, and G550W-CFTR channels showed increased PKA sensitivity and open probability. After treatment with ELX-02 and CFTR correctors, CFTR function rescued from the G550X allele in FRTs reached 20-40% of the wild-type level. These results suggest that readthrough of G550X produces greater CFTR function because of gain-of-function properties of the CFTR readthrough product that stem from its location in the signature LSGGQ motif found in ATP-binding cassette (ABC) transporters. G550X may be a particularly sensitive target for translational readthrough therapy.NEW & NOTEWORTHY We found that forskolin-induced swelling in G550X-CFTR patient-derived intestinal organoids (PDOs) was significantly higher than in G542X-CFTR PDOs after treatment with ELX-02. Tryptophan (W) was the sole amino acid inserted in the G550X position after readthrough. Resulting G550W-CFTR protein exhibited supernormal CFTR activity, PKA sensitivity, and open probability. These results show that aminoglycoside-induced readthrough of G550X produces greater CFTR function because of the gain-of-function properties of the CFTR readthrough product.

Ivacaftor Reverses Airway Mucus Abnormalities in a Rat Model Harboring a Humanized G551D-CFTR.

Rationale: Animal models have been highly informative for understanding the characteristics, onset, and progression of cystic fibrosis (CF) lung disease. In particular, the CFTR-/- rat has revealed insights into the airway mucus defect characteristic of CF but does not replicate a human-relevant CFTR (cystic fibrosis transmembrane conductance regulator) variant.Objectives: We hypothesized that a rat expressing a humanized version of CFTR and harboring the ivacaftor-sensitive variant G551D could be used to test the impact of CFTR modulators on pathophysiologic development and correction.Methods: In this study, we describe a humanized-CFTR rat expressing the G551D variant obtained by zinc finger nuclease editing of a human complementary DNA superexon, spanning exon 2-27, with a 5' insertion site into the rat gene just beyond intron 1. This targeted insertion takes advantage of the endogenous rat promoter, resulting in appropriate expression compared with wild-type animals.Measurements and Main Results: The bioelectric phenotype of the epithelia recapitulates the expected absence of CFTR activity, which was restored with ivacaftor. Large airway defects, including depleted airway surface liquid and periciliary layers, delayed mucus transport rates, and increased mucus viscosity, were normalized after the administration of ivacaftor.Conclusions: This model is useful to understand the mechanisms of disease and the extent of pathology reversal with CFTR modulators.

Novel Therapy of Bicarbonate, Glutathione, and Ascorbic Acid Improves Cystic Fibrosis Mucus Transport.

Defective airway mucus clearance is a defining characteristic of cystic fibrosis lung disease, and improvements to current mucolytic strategies are needed. Novel approaches targeting a range of contributing mechanisms are in various stages of preclinical and clinical development. ARINA-1 is a new nebulized product comprised of ascorbic acid, glutathione, and bicarbonate. Using microoptical coherence tomography, we tested the effect of ARINA-1 on central features of mucociliary clearance in F508del/F508del primary human bronchial epithelial cells to assess its potential as a mucoactive therapy in cystic fibrosis. We found that ARINA-1 significantly augmented mucociliary transport rates, both alone and with CFTR (cystic fibrosis transmembrane conductance regulator) modulator therapy, whereas airway hydration and ciliary beating were largely unchanged compared with PBS vehicle control. Analysis of mucus reflectivity and particle-tracking microrheology indicated that ARINA-1 restores mucus clearance by principally reducing mucus layer viscosity. The combination of bicarbonate and glutathione elicited increases in mucociliary transport rate comparable to those seen with ARINA-1, indicating the importance of this interaction to the impact of ARINA-1 on mucus transport; this effect was not recapitulated with bicarbonate alone or bicarbonate combined with ascorbic acid. Assessment of CFTR chloride transport revealed an increase in CFTR-mediated chloride secretion in response to ARINA-1 in CFBE41o- cells expressing wild-type CFTR, driven by CFTR activity stimulation by ascorbate. This response was absent in CFBE41o- F508del cells treated with VX-809 and primary human bronchial epithelial cells, implicating CFTR-independent mechanisms for the effect of ARINA-1 on cystic fibrosis mucus. Together, these studies indicate that ARINA-1 is a novel potential therapy for the treatment of impaired mucus clearance in cystic fibrosis.

G551D mutation impairs PKA-dependent activation of CFTR channel that can be restored by novel GOF mutations.

G551D is a major disease-associated gating mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an ATP- and phosphorylation-dependent chloride channel. G551D causes severe cystic fibrosis (CF) disease by disrupting ATP-dependent channel opening; however, whether G551D affects phosphorylation-dependent channel activation is unclear. Here, we use macropatch recording and Ussing chamber approaches to demonstrate that G551D impacts on phosphorylation-dependent activation of CFTR, and PKA-mediated phosphorylation regulates the interaction between the x-loop in nucleotide-binding domain 2 (NBD2) and cytosolic loop (CL) 1. We show that G551D not only disrupts ATP-dependent channel opening but also impairs phosphorylation-dependent channel activation by largely reducing PKA sensitivity consistent with the reciprocal relationship between channel opening/gating, ligand binding, and phosphorylation. Furthermore, we identified two novel GOF mutations: D1341R in the x-loop near the ATP-binding cassette signature motif in NBD2 and D173R in CL1, each of which strongly increased PKA sensitivity both in the wild-type (WT) background and when introduced into G551D-CFTR. When D1341R was combined with a second GOF mutation (e.g., K978C in CL3), we find that the double GOF mutation maximally increased G551D channel activity such that VX-770 had no further effect. We further show that a double charge-reversal mutation of D1341R/D173R-CFTR exhibited similar PKA sensitivity when compared with WT-CFTR. Together, our results suggest that charge repulsion between D173 and D1341 of WT-CFTR normally inhibits channel activation at low PKA activity by reducing PKA sensitivity, and negative allostery by the G551D is coupled to reduced PKA sensitivity of CFTR that can be restored by second GOF mutations.

Desmin loss and mitochondrial damage precede left ventricular systolic failure in volume overload heart failure.

Heart failure due to chronic volume overload (VO) in rats and humans is characterized by disorganization of the cardiomyocyte desmin/mitochondrial network. Here, we tested the hypothesis that desmin breakdown is an early and continuous process throughout VO. Male Sprague-Dawley rats had aortocaval fistula (ACF) or sham surgery and were examined 24 h and 4 and 12 wk later. Desmin/mitochondrial ultrastructure was examined by transmission electron microscopy (TEM) and immunohistochemistry (IHC). Protein and kinome analysis were performed in isolated cardiomyocytes, and desmin cleavage was assessed by mass spectrometry in left ventricular (LV) tissue. Echocardiography demonstrated a 40% decrease in the LV mass-to-volume ratio with spherical remodeling at 4 wk with ACF and LV systolic dysfunction at 12 wk. Starting at 24 h and continuing to 4 and 12 wk, with ACF there is TEM evidence of extensive mitochondrial clustering, IHC evidence of disorganization associated with desmin breakdown, and desmin protein cleavage verified by Western blot analysis and mass spectrometry. IHC results revealed that ACF cardiomyocytes at 4 and 12 wk had perinuclear translocation of αB-crystallin from the Z disk with increased α, ß-unsaturated aldehyde 4-hydroxynonelal. Use of protein markers with verification by TUNEL staining and kinome analysis revealed an absence of cardiomyocyte apoptosis at 4 and 12 wk of ACF. Significant increases in protein indicators of mitophagy were countered by a sixfold increase in p62/sequestosome-1, which is indicative of an inability to complete autophagy. An early and continuous disruption of the desmin/mitochondrial architecture, accompanied by oxidative stress and inhibition of apoptosis and mitophagy, suggests its causal role in LV dilatation and systolic dysfunction in VO.NEW & NOTEWORTHY This study provides new evidence of early onset (24 h) and continuous (4-12 wk) desmin misarrangement and disruption of the normal sarcomeric and mitochondrial architecture throughout the progression of volume overload heart failure, suggesting a causal link between desmin cleavage and mitochondrial disorganization and damage.

A synonymous codon change alters the drug sensitivity of ΔF508 cystic fibrosis transmembrane conductance regulator.

Synonymous mutations, such as I507-ATC→ATT, in deletion of Phe508 in cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR), the most frequent disease-associated mutant of CFTR, may affect protein biogenesis, structure, and function and contribute to an altered disease phenotype. Small-molecule drugs are being developed to correct ΔF508 CFTR. To understand correction mechanisms and the consequences of synonymous mutations, we analyzed the effect of mechanistically distinct correctors, corrector 4a (C4) and lumacaftor (VX-809), on I507-ATT and I507-ATC ΔF508 CFTR biogenesis and function. C4 stabilized I507-ATT ΔF508 CFTR band B, but without considerable biochemical and functional correction. VX-809 biochemically corrected ∼10% of both of the variants, leading to stable, forskolin+3-isobutyl-1-methylxanthine (IBMX)-activated whole-cell currents in the presence of the corrector. Omitting VX-809 during whole-cell recordings led to a spontaneous decline of the currents, suggesting posttranslational stabilization by VX-809. Treatment of cells with the C4+VX-809 combination resulted in enhanced rescue and 2-fold higher forskolin+IBMX-activated currents of both I507-ATT and I507-ATC ΔF508 CFTR, compared with VX-809 treatment alone. The lack of an effect of C4 on I507-ATC ΔF508 CFTR, but its additive effect in combination with VX-809, implies that C4 acted on VX-809-modified I507-ATC ΔF508 CFTR. Our results suggest that binding of C4 and VX-809 to ΔF508 CFTR is conformation specific and provide evidence that synonymous mutations can alter the drug sensitivity of proteins.

Increased fibroblast chymase production mediates procollagen autophagic digestion in volume overload.

BACKGROUND: Previous work has identified mast cells as the major source of chymase largely associated with a profibrotic phenotype. We recently reported increased fibroblast autophagic procollagen degradation in a rat model of pure volume overload (VO). Here we demonstrate a connection between increased fibroblast chymase production and autophagic digestion of procollagen in the pure VO of aortocaval fistula (ACF) in the rat. METHODS AND RESULTS: Isolated LV fibroblasts taken from 4 and 12week ACF Sprague-Dawley rats have significant increases in chymase mRNA and chymase activity. Increased intracellular chymase protein is documented by immunocytochemistry in the ACF fibroblasts compared to cells obtained from age-matched sham rats. To implicate VO as a stimulus for chymase production, we show that isolated adult rat LV fibroblasts subjected to 24h of 20% cyclical stretch induces chymase mRNA and protein production. Exogenous chymase treatment of control isolated adult cardiac fibroblasts demonstrates that chymase is internalized through a dynamin-dependent mechanism. Chymase treatment leads to an increased formation of autophagic vacuoles, LC3-II production, autophagic flux, resulting in increased procollagen degradation. Chymase inhibitor treatment reduces cyclical stretch-induced autophagy in isolated cardiac fibroblasts, demonstrating chymase's role in autophagy induction. CONCLUSION: In a pure VO model, chymase produced in adult cardiac fibroblasts leads to autophagic degradation of newly synthesized intracellular procollagen I, suggesting a new role of chymase in extracellular matrix degradation.

Codon bias and the folding dynamics of the cystic fibrosis transmembrane conductance regulator.

Synonymous or silent mutations are often overlooked in genetic analyses for disease-causing mutations unless they are directly associated with potential splicing defects. More recent studies, however, indicate that some synonymous single polynucleotide polymorphisms (sSNPs) are associated with changes in protein expression, and in some cases, protein folding and function. The impact of codon usage and mRNA structural changes on protein translation rates and how they can affect protein structure and function is just beginning to be appreciated. Examples are given here that demonstrate how synonymous mutations alter the translational kinetics and protein folding and/or function. The mechanism for how this occurs is based on a model in which codon usage modulates the translational rate by introducing pauses caused by nonoptimal or rare codons or by introducing changes in the mRNA structure, and this in turn influences co-translational folding. Two examples of this include the multidrug resistance protein (p-glycoprotein) and the cystic fibrosis transmembrane conductance regulator gene (CFTR). CFTR is also used here as a model to illustrate how synonymous mutations can be examined using in silico predictive methods to identify which sSNPs have the potential to change protein structure. The methodology described here can be used to help identify "non-silent" synonymous mutations in other genes.

Volume overload induces autophagic degradation of procollagen in cardiac fibroblasts.

In a pure volume overloaded (VO) heart, interstitial collagen loss is degraded by matrix metalloproteinases (MMPs) that leads to left ventricular (LV) dilatation and heart failure. Cardiac fibroblasts are the primary source of extracellular matrix proteins that connect cardiomyocytes. The goal of this study was to determine how VO affects intracellular procollagen in cardiac fibroblasts. Using the aortocaval fistula (ACF) model in Sprague-Dawley rats, we demonstrate that cardiac fibroblasts isolated from 4 and 12 wk ACF animals have decreased intracellular procollagen I compared to the fibroblasts from age-matched shams. The reduction of procollagen I is associated with increased autophagy as demonstrated by increased autophagic vacuoles and LC3-II expression. To test the relationship between autophagy and procollagen degradation, we treated adult cardiac fibroblasts with either an autophagy inducer, rapamycin, or an inhibitor, wortmannin, and found that procollagen I protein levels were decreased in fibroblasts treated with rapamycin and elevated in wortmannin-treated cells. In addition, we demonstrated that VO induces oxidative stresses in cardiac fibroblasts from 4 and 12 wk ACF rats. Treatment of cultured cardiac fibroblasts with an oxidative stress-inducing agent (DMNQ) induces autophagy and intracellular procollagen I and fibronectin degradation, which is reversed by wortmannin but not by the global MMP inhibitor (PD166793). Mechanical stretch of cardiac fibroblasts also induces oxidative stress and autophagic degradation of procollagen I and fibronectin. Our results suggest that in addition to the well-known effects of MMPs on extracellular collagen degradation in VO, there is a concurrent degradation of intracellular procollagen and fibronectin mediated by oxidative stress-induced autophagy in cardiac fibroblasts.

Rescuing ΔF508 CFTR with trimethylangelicin, a dual-acting corrector and potentiator.

Since the discovery of the cystic fibrosis (CF) gene that encodes the CF transmembrane conductance regulator (CFTR) in 1989, there has been considerable progress in understanding the molecular defects associated with different mutations in the CFTR protein. Small molecule "potentiators" have led the way as a drug therapeutic approach for correcting channel gating mutations such as the G551D mutation. Therapies for correcting the most common folding mutation in CFTR, ΔF508, however, have proven to be much more challenging. The protein-folding problem appears to be associated with both nucleotide binding domain (NBD) instability and domain interface interactions that are caused by the loss of the phenylalanine residue in NBD 1. Given the inherent complexity in the sequential folding pathway for this very large multidomain protein, it has been suggested that correcting the proper folding, anion channel function, and cell surface stability of the ΔF508 CFTR protein will require a multidrug approach to fix each of these compounding problems. Here we discuss a recent publication (Favia M, Mancini MT, Bezzerri V, Guerra L, Laselva O, Abbattiscianni AC, Debellis L, Reshkin SJ, Gambari R, Cabrini G, Casavola V. Am J Physiol Lung Cell Mol Physiol 307: L48-L61, 2014), however, that offers hope that single drug therapies are still possible.

The silent codon change I507-ATC->ATT contributes to the severity of the ΔF508 CFTR channel dysfunction.

The most common disease-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is the out-of-frame deletion of 3 nucleotides (CTT). This mutation leads to the loss of phenylalanine-508 (ΔF508) and a silent codon change (SCC) for isoleucine-507 (I507-ATC→ATT). ΔF508 CFTR is misfolded and degraded by endoplasmic reticulum-associated degradation (ERAD). We have demonstrated that the I507-ATC→ATT SCC alters ΔF508 CFTR mRNA structure and translation dynamics. By comparing the biochemical and functional properties of the I507-ATT and I507-ATC ΔF508 CFTR, we establish that the I507-ATC→ATT SCC contributes to the cotranslational misfolding, ERAD, and to the functional defects associated with ΔF508 CFTR. We demonstrate that the I507-ATC ΔF508 CFTR is less susceptible to the ER quality-control machinery during translation than the I507-ATT, although 27°C correction is necessary for sufficient cell-surface expression. Whole-cell patch-clamp recordings indicate sustained, thermally stable cAMP-activated Cl(-) transport through I507-ATC and unstable function of the I507-ATT ΔF508 CFTR. Single-channel recordings reveal improved gating properties of the I507-ATC compared to I507-ATT ΔF508 CFTR (NPo=0.45±0.037 vs. NPo=0.09±0.002; P<0.001). Our results signify the role of the I507-ATC→ATT SCC in the ΔF508 CFTR defects and support the importance of synonymous codon choices in determining the function of gene products.

Extended stop codon context predicts nonsense codon readthrough efficiency in human cells.

Protein synthesis terminates when a stop codon enters the ribosome's A-site. Although termination is efficient, stop codon readthrough can occur when a near-cognate tRNA outcompetes release factors during decoding. Seeking to understand readthrough regulation we used a machine learning approach to analyze readthrough efficiency data from published HEK293T ribosome profiling experiments and compared it to comparable yeast experiments. We obtained evidence for the conservation of identities of the stop codon, its context, and 3'-UTR length (when termination is compromised), but not the P-site codon, suggesting a P-site tRNA role in readthrough regulation. Models trained on data from cells treated with the readthrough-promoting drug, G418, accurately predicted readthrough of premature termination codons arising from CFTR nonsense alleles that cause cystic fibrosis. This predictive ability has the potential to aid development of nonsense suppression therapies by predicting a patient's likelihood of improvement in response to drugs given their nonsense mutation sequence context.

Dab2 is a key regulator of endocytosis and post-endocytic trafficking of the cystic fibrosis transmembrane conductance regulator.

CFTR (cystic fibrosis transmembrane conductance regulator) is expressed in the apical membrane of epithelial cells. Cell-surface CFTR levels are regulated by endocytosis and recycling. A number of adaptor proteins including AP-2 (µ2 subunit) and Dab2 (Disabled-2) have been proposed to modulate CFTR internalization. In the present study we have used siRNA (small interfering RNA)-mediated silencing of these adaptors to test their roles in the regulation of CFTR cell-surface trafficking and stability in human airway epithelial cells. The results indicate that µ2 and Dab2 performed partially overlapping, but divergent, functions. While µ2 depletion dramatically decreased CFTR endocytosis with little effect on the half-life of the CFTR protein, Dab2 depletion increased the CFTR half-life ~3-fold, in addition to inhibiting CFTR endocytosis. Furthermore, Dab2 depletion inhibited CFTR trafficking from the sorting endosome to the recycling compartment, as well as delivery of CFTR to the late endosome, thus providing a mechanistic explanation for increased CFTR expression and half-life. To test whether two E3 ligases were required for the endocytosis and/or down-regulation of surface CFTR, we siRNA-depleted CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein] and c-Cbl (casitas B-lineage lymphoma). We demonstrate that CHIP and c-Cbl depletion have no effect on CFTR endocytosis, but c-Cbl depletion modestly enhanced the half-life of CFTR. The results of the present study define a significant role for Dab2 both in the endocytosis and post-endocytic fate of CFTR.

RNA binding proteins PTBP1 and HNRNPL regulate CFTR mRNA decay.

Background: CFTR nonsense alleles generate negligible CFTR protein due to the nonsense mutation: 1) triggering CFTR mRNA degradation by nonsense-mediated mRNA decay (NMD), and 2) terminating CFTR mRNA translation prematurely. Thus, people with cystic fibrosis (PwCF) who carry nonsense alleles cannot benefit from current modulator drugs, which target CFTR protein. In this study, we examined whether PTBP1 and HNRNPL, two RNA binding proteins that protect a subset of mRNAs with a long 3' untranslated region (UTR) from NMD, similarly affect CFTR mRNA.Silencing RNAs were used to deplete PTBP1 or HNRNPL in 16HBE14o- human bronchial epithelial cells expressing WT, G542X, or W1282X CFTR. CFTR mRNA abundance was measured relative to controls by quantitative PCR. PTBP1 and HNRNPL were also exogenously expressed in each cell line and CFTR mRNA levels were similarly quantified. Results: PTBP1 depletion reduced CFTR mRNA abundance in all three 16HBE14o- cell lines; HRNPL depletion reduced CFTR mRNA abundance in only the G542X and W1282X cell lines. Notably, decreased CFTR mRNA abundance correlated with increased mRNA decay. Exogenous expression of PTBP1 or HNRNPL increased CFTR mRNA abundance in all three cell lines; HNRNPL overexpression generally increased CFTR to a greater extent in G542X and W1282X 16HBE14o- cells.Our data indicate that PTBP1 and HNRNPL regulate CFTR mRNA abundance by protecting CFTR transcripts from NMD. This suggests that PTBP1 and/or HNRNPL may represent potential therapeutic targets to increase CFTR mRNA abundance and enhance responses to CFTR modulators and other therapeutic approaches in PwCF.

Prime editing-mediated correction of the CFTR W1282X mutation in iPSCs and derived airway epithelial cells.

A major unmet need in the cystic fibrosis (CF) therapeutic landscape is the lack of effective treatments for nonsense CFTR mutations, which affect approximately 10% of CF patients. Correction of nonsense CFTR mutations via genomic editing represents a promising therapeutic approach. In this study, we tested whether prime editing, a novel CRISPR-based genomic editing method, can be a potential therapeutic modality to correct nonsense CFTR mutations. We generated iPSCs from a CF patient homozygous for the CFTR W1282X mutation. We demonstrated that prime editing corrected one mutant allele in iPSCs, which effectively restored CFTR function in iPSC-derived airway epithelial cells and organoids. We further demonstrated that prime editing may directly repair mutations in iPSC-derived airway epithelial cells when the prime editing machinery is efficiently delivered by helper-dependent adenovirus (HDAd). Together, our data demonstrated that prime editing may potentially be applied to correct CFTR mutations such as W1282X.

Mucociliary transport deficiency and disease progression in Syrian hamsters with SARS-CoV-2 infection.

Substantial clinical evidence supports the notion that ciliary function in the airways is important in COVID-19 pathogenesis. Although ciliary damage has been observed in both in vitro and in vivo models, the extent or nature of impairment of mucociliary transport (MCT) in in vivo models remains unknown. We hypothesize that SARS-CoV-2 infection results in MCT deficiency in the airways of golden Syrian hamsters that precedes pathological injury in lung parenchyma. Micro-optical coherence tomography was used to quantitate functional changes in the MCT apparatus. Both genomic and subgenomic viral RNA pathological and physiological changes were monitored in parallel. We show that SARS-CoV-2 infection caused a 67% decrease in MCT rate as early as 2 days postinfection (dpi) in hamsters, principally due to 79% diminished airway coverage of motile cilia. Correlating quantitation of physiological, virological, and pathological changes reveals steadily descending infection from the upper airways to lower airways to lung parenchyma within 7 dpi. Our results indicate that functional deficits of the MCT apparatus are a key aspect of COVID-19 pathogenesis, may extend viral retention, and could pose a risk factor for secondary infection. Clinically, monitoring abnormal ciliated cell function may indicate disease progression. Therapies directed toward the MCT apparatus deserve further investigation.

Mucociliary Transport Deficiency and Disease Progression in Syrian Hamsters with SARS-CoV-2 Infection.

Substantial clinical evidence supports the notion that ciliary function in the airways plays an important role in COVID-19 pathogenesis. Although ciliary damage has been observed in both in vitro and in vivo models, consequent impaired mucociliary transport (MCT) remains unknown for the intact MCT apparatus from an in vivo model of disease. Using golden Syrian hamsters, a common animal model that recapitulates human COVID-19, we quantitatively followed the time course of physiological, virological, and pathological changes upon SARS-CoV-2 infection, as well as the deficiency of the MCT apparatus using micro-optical coherence tomography, a novel method to visualize and simultaneously quantitate multiple aspects of the functional microanatomy of intact airways. Corresponding to progressive weight loss up to 7 days post-infection (dpi), viral detection and histopathological analysis in both the trachea and lung revealed steadily descending infection from the upper airways, as the main target of viral invasion, to lower airways and parenchymal lung, which are likely injured through indirect mechanisms. SARS-CoV-2 infection caused a 67% decrease in MCT rate as early as 2 dpi, largely due to diminished motile ciliation coverage, but not airway surface liquid depth, periciliary liquid depth, or cilia beat frequency of residual motile cilia. Further analysis indicated that the fewer motile cilia combined with abnormal ciliary motion of residual cilia contributed to the delayed MCT. The time course of physiological, virological, and pathological progression suggest that functional deficits of the MCT apparatus predispose to COVID-19 pathogenesis by extending viral retention and may be a risk factor for secondary infection. As a consequence, therapies directed towards the MCT apparatus deserve further investigation as a treatment modality.

Human cytomegalovirus UL97 kinase prevents the deposition of mutant protein aggregates in cellular models of Huntington's disease and ataxia.

The presence of aggregates of abnormally expanded polyglutamine (polyQ)-containing proteins are a pathological hallmark of a number of neurodegenerative diseases including Huntington's disease (HD) and spinocerebellar ataxia-3 (SCA3). Previous studies in cellular, Drosophila, and mouse models of HD and SCA have shown that neurodegeneration can be prevented by manipulations that inhibit polyQ aggregation. We have shown that the UL97 kinase of the human cytomegalovirus (HCMV) prevents aggregation of the pp71 and pp65 viral tegument proteins. To explore whether UL97 may act as a general antiaggregation factor, we examined whether UL97 prevents aggregation of cellular non-polyQ and polyQ proteins. We report that UL97 prevents the deposition of aggregates of two non-polyQ proteins: a protein chimera (GFP170*) composed of the green fluorescent protein and a fragment of the Golgi Complex protein (GCP-170) and a chimera composed of the red fluorescent protein (RFP) fused to the Werner syndrome protein (WRN), a RecQ helicase and exonuclease involved in DNA repair. Furthermore, we show that UL97 inhibits aggregate deposition in cellular models of HD and SCA3. UL97 prevents the deposition of aggregates of the mutant huntingtin exon 1 containing 82 glutamine repeats (HttExon1-Q82) or full length ataxin-3 containing a 72 polyQ track (AT3-72Q). The kinase activity of UL97 appears critical, as the kinase-dead UL97 mutant (K335M) fails to prevent aggregate formation. We further show that UL97 disrupts nuclear PML bodies and decreases p53-mediated transcription. The universality of the antiaggregation effect of UL97 suggests that UL97 targets a key cellular factor that regulates cellular aggregation mechanisms. Our results identify UL97 as a novel means to modulate polyQ aggregation and suggest that UL97 can serve as a novel tool to probe the cellular mechanisms that contribute to the formation of aggregates in polyglutamine disorders.

A small molecule that induces translational readthrough of CFTR nonsense mutations by eRF1 depletion.

Premature termination codons (PTCs) prevent translation of a full-length protein and trigger nonsense-mediated mRNA decay (NMD). Nonsense suppression (also termed readthrough) therapy restores protein function by selectively suppressing translation termination at PTCs. Poor efficacy of current readthrough agents prompted us to search for better compounds. An NMD-sensitive NanoLuc readthrough reporter was used to screen 771,345 compounds. Among the 180 compounds identified with readthrough activity, SRI-37240 and its more potent derivative SRI-41315, induce a prolonged pause at stop codons and suppress PTCs associated with cystic fibrosis in immortalized and primary human bronchial epithelial cells, restoring CFTR expression and function. SRI-41315 suppresses PTCs by reducing the abundance of the termination factor eRF1. SRI-41315 also potentiates aminoglycoside-mediated readthrough, leading to synergistic increases in CFTR activity. Combining readthrough agents that target distinct components of the translation machinery is a promising treatment strategy for diseases caused by PTCs.

Targets for cystic fibrosis therapy: proteomic analysis and correction of mutant cystic fibrosis transmembrane conductance regulator.

Proteomic analysis has proved to be an important tool for understanding the complex nature of genetic disorders, such as cystic fibrosis (CF), by defining the cellular protein environment (proteome) associated with wild-type and mutant proteins. Proteomic screens identified the proteome of CF transmembrane conductance regulator (CFTR), and provided fundamental information to studies designed for understanding the crucial components of physiological CFTR function. Simultaneously, high-throughput screens for small-molecular correctors of CFTR mutants provided promising candidates for therapy. The majority of CF cases are caused by nucleotide deletions (DeltaF508 CFTR; >75%), resulting in CFTR misfolding, or insertion of premature termination codons ( approximately 10%), leading to unstable mRNA and reduced levels of truncated dysfunctional CFTR. In this article, we review recent results of proteomic screens, developments in identifying correctors for the most frequent CFTR mutants, and comment on how integration of the knowledge gained from these studies may aid in finding a cure for CF and a number of other genetic disorders.