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RPL12 knockdown is additive with VX-809 in primary CF human bronchial epithelia (HBE). (A) The effect of RPL12 knockdown, VX-809 treatment (3 μM, 24 h) or combination of both on the expression pattern of endogenous ΔF508-CFTR in HBE cells isolated from one CF patient with CFTRΔF508/ΔF508 genotype (upper panel, patient code 21). Immunoblots were probed with antibodies against CFTR (1:1 mixture of UNC antibodies 570 and 596), Rpl12, and β-actin as a loading control. Expression levels of Rpl12 and the core- (band B, filled arrowhead) or complex-glycosylated (band C, open arrowhead) ΔF508-CFTR were quantified by densitometry and are depicted as a percentage compared to controls transfected with NT siRNA (lower panel, n = 3). (B) Representative Isc recordings (upper panel) and quantification of the changes (ΔIsc, lower panel) after siRNA-mediated RPL12 knockdown, NT siRNA or mock transfection in HBE cells homozygous for ΔF508-CFTR CFTR (patient code 21, n = 3). A representative Isc trace of HBE with CFTRWT/WT genotype is shown for comparison (upper panel). (C) Quantification of the Frk- and gen-stimulated current (ΔIsc Frk + gen) in HBE isolated from three different homozygous ΔF508 CF patients (21, 22, and 48) or one WT-CFTR donor (10) transfected with RPL12_6 or NT siRNA for 21 days alone or in combination with VX-809 (3 μM, 24 h) (C) and VX-661 (3 μM, 24 h). CFTR mediated currents were induced by sequential acute addition of Frk (10 μM) and gen (50 μM) followed by CFTR inhibition with inhibitor 172 (Inh172, 10 μM) in the presence of a basolateral-to-apical chloride gradient. *p < 0.05; **p < 0.01; ***p < 0.001. Error bars show SEM of three independent experiments. The underlying data of panels A–C can be found in S1 Data.
Source publication
The most common cystic fibrosis (CF) causing mutation, deletion of phenylalanine 508 (ΔF508 or Phe508del), results in functional expression defect of the CF transmembrane conductance regulator (CFTR) at the apical plasma membrane (PM) of secretory epithelia, which is attributed to the degradation of the misfolded channel at the endoplasmic reticulu...
Citations
... Our study suggests that therapeutic approaches that reduce molecular crowding by alleviating ER stress through reduced protein misfolding or protein synthesis and/or enhancing degradation of the unfolded proteins may improve mucus properties 29,40 . Reducing the rate of protein synthesis also improves the folding of deltaF508-CFTR 41,42 . ...
Abnormally viscous and thick mucus is a hallmark of cystic fibrosis (CF). How the genetic defect causes abnormal mucus in CF remains unanswered and a question of paramount interest. Mucus is produced by hydration of gel-forming mucin macromolecules that are stored in secretory granules prior to release. Current understanding of mucin/mucus structure before and after secretion remains limited and contradictory models exist. Here we used a molecular viscometer and fluorescence lifetime imaging of primary epithelial cells (Normal and CF) to measure nanometer-scale viscosity. We found significantly elevated intraluminal nanoviscosity in a population of CF mucin granules, indicating an intrinsic, pre-secretory, mucin defect. Validation experiments showed that high nanoviscosity in cellular environments is mainly due to the low mobility of water that hydrates macromolecules. Nanoviscosity influences protein conformational dynamics and function. Its elevation along the protein secretory pathway indicates molecular overcrowding and is expected to alter mucins post-translational processing, hydration, and mucus rheology after release. The nanoviscosity of extracellular CF mucus was elevated compared to non-CF mucus. Remarkably, it was higher after secretion than in granules, which suggests mucins have a weakly-ordered state in granules and adopt a highly-ordered, nematic crystalline structure extracellularly. This challenges the classical view of mucus as a porous agarose-like gel and suggests an alternative model for mucin organization before and after secretion. Our study also suggests that endoplasmic reticulum stress due to molecular overcrowding contributes to mucus pathogenesis in CF cells. It encourages the development of therapeutics that target pre-secretory mechanisms in CF and other muco-obstructive lung diseases.
... Interestingly, KD of eIF3a, a subunit of the eukaryotic initiation factor 3, rescued CFTR folding in various mutants by slowing translation (55). Blunting translational rate by altering ribosomal velocity via RPL12 KD has been shown to rescue F508del maturation (49,56). Moreover, early cotranslational misfolding of P67L may result in ribosome stalling and trigger ribosomal quality control to terminate translation (57). ...
Cystic fibrosis (CF) is one of the most prevalent lethal genetic diseases with over 2000 identified mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Pharmacological chaperones such as Lumacaftor (VX-809), Tezacaftor (VX-661) and Elexacaftor (VX-445) treat mutation-induced defects by stabilizing CFTR and are called correctors. These correctors improve proper folding and thus facilitate processing and trafficking to increase the amount of functional CFTR on the cell surface. Yet, CFTR variants display differential responses to each corrector. Here, we report variants P67L and L206W respond similarly to VX-809 but divergently to VX-445 with P67L exhibiting little rescue when treated with VX-445. We investigate the underlying cellular mechanisms of how CFTR biogenesis is altered by correctors in these variants. Affinity purification-mass spectrometry (AP-MS) multiplexed with isobaric Tandem Mass Tags (TMT) was used to quantify CFTR protein-protein interaction changes between variants P67L and L206W. VX-445 facilitates unique proteostasis factor interactions especially in translation, folding, and degradation pathways in a CFTR variant-dependent manner. A number of these interacting proteins knocked down by siRNA, such as ribosomal subunit proteins, moderately rescued fully glycosylated P67L. Importantly, these knockdowns sensitize P67L to VX-445 and further enhance the trafficking correction of this variant. Partial inhibition of protein translation also mildly sensitizes P67L CFTR to VX-445 correction, supporting a role for translational dynamics in the rescue mechanism of VX-445. Our results provide a better understanding of VX-445 biological mechanism of action and reveal cellular targets that may sensitize unresponsive CFTR variants to known and available correctors.
... Approximately 50% reduction in uL11 expression attenuates translation initiation and elongation . Findings also indicate uL11 depletion enhances protein maturation/stability and transepithelial ion transport exhibited by p.Phe508del and other CFTR missense variants (Veit et al., 2016;Oliver et al., 2019). Interestingly, siRNAmediated knockdown of eukaryotic initiation factor 3a also improves p.Phe508del protein trafficking and function (Hutt et al., 2018). ...
Cystic fibrosis (CF) is an autosomal recessive disease impacting ∼100,000 people worldwide. This lethal disorder is caused by mutation of the CF transmembrane conductance regulator (CFTR) gene, which encodes an ATP-binding cassette-class C protein. More than 2,100 variants have been identified throughout the length of CFTR. These defects confer differing levels of severity in mRNA and/or protein synthesis, folding, gating, and turnover. Drug discovery efforts have resulted in recent development of modulator therapies that improve clinical outcomes for people living with CF. However, a significant portion of the CF population has demonstrated either no response and/or adverse reactions to small molecules. Additional therapeutic options are needed to restore underlying genetic defects for all patients, particularly individuals carrying rare or refractory CFTR variants. Concerted focus has been placed on rescuing variants that encode truncated CFTR protein, which also harbor abnormalities in mRNA synthesis and stability. The current mini-review provides an overview of CFTR mRNA features known to elicit functional consequences on final protein conformation and function, including considerations for RNA-directed therapies under investigation. Alternative exon usage in the 5′-untranslated region, polypyrimidine tracts, and other sequence elements that influence splicing are discussed. Additionally, we describe mechanisms of CFTR mRNA decay and post-transcriptional regulation mediated through interactions with the 3′-untranslated region (e.g. poly-uracil sequences, microRNAs). Contributions of synonymous single nucleotide polymorphisms to CFTR transcript utilization are also examined. Comprehensive understanding of CFTR RNA biology will be imperative for optimizing future therapeutic endeavors intended to address presently untreatable forms of CF.
... It was demonstrated that the translation rate acts as a substantial contributor to CFTR functional expression [31][32][33]. Indeed, slowing down the rates of translation initiation and elongation enhances WT-CFTR biogenesis (and, therefore, its cell surface expression and function) by improving its folding [31]. The slowing down of the translational rate to improve CFTR expression could be achieved pharmacologically by treating the cells with a submaximal concentration of cycloheximide (CHX) to block protein synthesis [33] partially or by silencing the proteins of the ribosomal stalk [31,32]. ...
... Indeed, slowing down the rates of translation initiation and elongation enhances WT-CFTR biogenesis (and, therefore, its cell surface expression and function) by improving its folding [31]. The slowing down of the translational rate to improve CFTR expression could be achieved pharmacologically by treating the cells with a submaximal concentration of cycloheximide (CHX) to block protein synthesis [33] partially or by silencing the proteins of the ribosomal stalk [31,32]. Thus, we verified the effect of a translational rate slowdown on WT and [G576A;R668C] CFTR. ...
Carriers of single pathogenic variants of the CFTR (cystic fibrosis transmembrane conductance regulator) gene have a higher risk of severe COVID-19 and 14-day death. The machine learning post-Mendelian model pinpointed CFTR as a bidirectional modulator of COVID-19 outcomes. Here, we demonstrate that the rare complex allele [G576V;R668C] is associated with a milder disease via a gain-of-function mechanism. Conversely, CFTR ultra-rare alleles with reduced function are associated with disease severity either alone (dominant disorder) or with another hypomorphic allele in the second chromosome (recessive disorder) with a global residual CFTR activity between 50 to 91%. Furthermore, we characterized novel CFTR complex alleles, including [A238V;F508del], [R74W;D1270N;V201M], [I1027T;F508del], [I506V;D1168G], and simple alleles, including R347C, F1052V, Y625N, I328V, K68E, A309D, A252T, G542*, V562I, R1066H, I506V, I807M, which lead to a reduced CFTR function and thus, to more severe COVID-19. In conclusion, CFTR genetic analysis is an important tool in identifying patients at risk of severe COVID-19.
... In brief, the protein composition of CF sweat highlighted that factors of CF severity (CFTR genotype) can be monitored by sweat proteomics. From a pathophysiological perspective, ribosomal stalk proteins were described as modifiers of CF severity when the silencing of corresponding genes elicited the partial phenotype rescue of F508del CFTR processing defects [25]. Here, ribosomal stalk proteins uL11, P0 (uL10), and P2 plus ribosomal proteins uL4 and eL6 were sweat markers of CF disease and severity, respectively. ...
In clinical routine, the diagnosis of cystic fibrosis (CF) is still challenging regardless of international consensus on diagnosis guidelines and tests. For decades, the classical Gibson and Cooke test measuring sweat chloride concentration has been a keystone, yet, it may provide normal or equivocal results. As of now, despite the combination of sweat testing, CFTR genotyping, and CFTR functional testing, a small fraction (1–2%) of inconclusive diagnoses are reported and justifies the search for new CF biomarkers. More importantly, in the context of precision medicine, with a view to early diagnosis, better prognosis, appropriate clinical follow-up, and new therapeutic development, discovering companion biomarkers of CF severity and phenotypic rescue are of utmost interest. To date, previous sweat proteomic studies have already documented disease-specific variations of sweat proteins (e.g., in schizophrenia and tuberculosis). In the current study, sweat samples from 28 healthy control subjects and 14 patients with CF were analyzed by nanoUHPLC-Q-Orbitrap-based shotgun proteomics, to look for CF-associated changes in sweat protein composition and abundance. A total of 1057 proteins were identified and quantified at an individual level, by a shotgun label-free approach. Notwithstanding similar proteome composition, enrichment, and functional annotations, control and CF samples featured distinct quantitative proteome profiles significantly correlated with CF, accounting for the respective inter-individual variabilities of control and CF sweat. All in all: (i) 402 sweat proteins were differentially abundant between controls and patients with CF, (ii) 68 proteins varied in abundance between F508del homozygous patients and patients with another genotype, (iii) 71 proteins were differentially abundant according to the pancreatic function, and iv) 54 proteins changed in abundance depending on the lung function. The functional annotation of pathophysiological biomarkers highlighted eccrine gland cell perturbations in: (i) protein biosynthesis and trafficking, (ii) CFTR proteostasis and membrane stability, and (iii) cell-cell adherence, membrane integrity, and cytoskeleton crosstalk. Cytoskeleton-related biomarkers were of utmost interest because of the consistency between variations observed here in CF sweat and variations previously documented in other CF tissues. From a clinical stance, nine candidate biomarkers of CF diagnosis (CUTA, ARG1, EZR, AGA, FLNA, MAN1A1, MIA3, LFNG, SIAE) and seven candidate biomarkers of CF severity (ARG1, GPT, MDH2, EML4 (F508del homozygous), MGAT1 (pancreatic insufficiency), IGJ, TOLLIP (lung function impairment)) were deemed suitable for further verification.
... In certain cases, however, it might suffice to modulate the translation process in the ER to overcome CFTR defects. By reducing ribosome velocity during translation for example through suppression of the ribosomal protein L12 (RPL12), mutant CFTR trafficking and function has been shown to be partially restored [245,246]. ...
Cystic fibrosis (CF) is the most common monogenic disorder, caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Over the last 30 years, tremendous progress has been made in understanding the molecular basis of CF and the development of treatments that target the underlying defects in CF. Currently, a highly effective CFTR modulator treatment (Kalydeco™/Trikafta™) is available for 90% of people with CF. In this review, we will give an extensive overview of past and ongoing efforts in the development of therapies targeting the molecular defects in CF. We will discuss strategies targeting the CFTR protein (i.e., CFTR modulators such as correctors and potentiators), its cellular environment (i.e., proteostasis modulation, stabilization at the plasma membrane), the CFTR mRNA (i.e., amplifiers, nonsense mediated mRNA decay suppressors, translational readthrough inducing drugs) or the CFTR gene (gene therapies). Finally, we will focus on how these efforts can be applied to the 15% of people with CF for whom no causal therapy is available yet.
... We used CFTR as a prototypical example of the impact of variation on protein fold and function trajectories in response to the environment given the extensive number of variants contributing to disease in the CF population, but whose roles in disease remain largely mechanistically and clinically uncharacterized 27 . Export of F508del from the ER in response to reduced temperature is causally coupled to the co-translational program that, in response to the reduced rate of CFTR synthesis at 27°C 46,[78][79][80] , has been shown to provide a biological thermodynamic advantage to its folding 34,71,78,81 . We have now extended this observation to every residue in the polypeptide chain. ...
Although the impact of genome variation on the thermodynamic properties of function on the protein fold has been studied in vitro, it remains a challenge to assign these relationships across the entire polypeptide sequence in vivo. Using the Gaussian process regression based principle of Spatial CoVariance, we globally assign on a residue-by-residue basis the biological thermodynamic properties that contribute to the functional fold of CFTR in the cell. We demonstrate the existence of a thermodynamically sensitive region of the CFTR fold involving the interface between NBD1 and ICL4 that contributes to its export from endoplasmic reticulum. At the cell surface a new set of residues contribute uniquely to the management of channel function. These results support a general ‘quality assurance’ view of global protein fold management as an SCV principle describing the differential pre- and post-ER residue interactions contributing to compartmentalization of the energetics of the protein fold for function. Our results set the stage for future analyses of the quality systems managing protein sequence-to-function-to-structure broadly encompassing genome design leading to protein function in complex cellular relationships responsible for diversity and fitness in biology in response to the environment.
... Moreover, RPs are involved in the lifecycle of diverse viruses, for instance, subjecting phosphorylation to respond viral infection, interacting with viral proteins to facilitate replication, and hijacked by viral IRES to orchestrate translation [16][17][18][19]. RPLP1 is one of the elements of the lateral stalk in the 60S ribosomal subunit [20,21] and usually helps to orchestrate the elongation phase of translation [22,23]. Previous studies suggested that RPLP1 is a pivotal host factor that facilitates viral proteins synthesis of Zika virus (ZIKV), dengue viruses (DENV), and yellow fever virus (YFV) [24,25]. ...
Classical swine fever virus (CSFV), the etiological agent of classical swine fever (CSF), causes serious financial losses to the pig industry. Using yeast two-hybrid screening, we have previously identified ribosomal protein RPLP1 as a potential binding partner of CSFV NS4B. In this study, the interaction between host RPLP1 and CSFV NS4B was further characterized by co-immunoprecipitation (co-IP), glutathione S-transferase (GST) pulldown, and confocal microscopy. In addition, lentivirus-mediated shRNA knockdown of RPLP1 drastically attenuated CSFV growth, while stable overexpression of RPLP1 markedly enhanced CSFV production. Moreover, cellular RPLP1 expression was found to be significantly up-regulated along with CSFV infection. Dual-luciferase reporter assay showed that depletion of RPLP1 had no effects on the activity of CSFV internal ribosome entry site (IRES). In the first life cycle of CSFV, further studies revealed that RPLP1 depletion did not influence the intracellular viral RNA abundance but diminished the intracellular and extracellular progeny virus titers as well as the viral E2 protein expression, which indicates that RPLP1 is crucial for CSFV genome translation. In summary, this study demonstrated that RPLP1 interacts with CSFV NS4B and enhances virus production via promoting translation of viral genome.
... As Lukacs and colleagues previously identified RPL12 as a CFTR modifier, we used knockdown of RPL12 as an additional positive control. 24 When compared to RPL12 knockdown, cells treated with the RPL32 DsiRNAs showed an increase in peak chloride current of 54.6%. ...
... Interestingly, Lukacs and colleagues identified a component of the ribosomal stalk, RPL12, as an effector of ΔF508-CFTR. 24 Silencing of RPL12 attenuated the translational elongation rate and increased the folding efficiency and conformational stability of ΔF508-CFTR. Additionally, knockdown of RPL12 in combination with VX-809 restored ΔF508-CFTR function to ~50% of the wild-type channel in primary human airway epithelial cells. ...
Cystic fibrosis is a lethal autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The common ΔF508‐CFTR mutation results in protein misfolding and proteasomal degradation. If ΔF508‐CFTR trafficks to the cell surface, its anion channel function may be partially restored. Several in vitro strategies can partially correct ΔF508‐CFTR trafficking and function, including low‐temperature, small molecules, overexpression of miR‐138, or knockdown of SIN3A. The challenge remains to translate such interventions into therapies and to understand their mechanisms. One approach for connecting such interventions to small molecule therapies that has previously succeeded for cystic fibrosis and other diseases is via mRNA expression profiling and iterative searches of small molecules with similar expression signatures. Here, we query the Library of Integrated Network‐based Cellular Signatures (LINCS) using transcriptomic signatures from previously generated CF expression data, including RNAi‐ and low temperature‐based rescue signatures. This LINCS in silico screen prioritized 135 small molecules that mimicked our rescue interventions based on their genome‐wide transcriptional perturbations. Functional screens of these small molecules identified eight compounds that partially restored ΔF508‐CFTR function, as assessed by cAMP‐activated chloride conductance. Of these, XL147 rescued ΔF508‐CFTR function in primary CF airway epithelia, while also showing cooperativity when administered with C18. Improved CF corrector therapies are greatly needed and this integrative drug prioritization approach offers a novel method to both identify small molecules that may rescue ΔF508‐CFTR function and identify gene networks underlying such rescue.
... Due to the known interactions between CFTR and several E3 ubiquitin ligases, we chose to investigate ASB6 and ASB13 (components of the SOCS-box ubiquitin ligase complex), FBXO46 (F-box), ZFAND5 (zf-A20), and the BTB domain-containing proteins GZF1, KLHL29, and ZBTB38 [19,36,37,[48][49][50][51][52][53][54][55][56][57][58][59][60][61][62]. As Lukacs and colleagues recently identified several components of the ribosomal stalk as being CFTR effectors, we also elected to study RPL15, RPL28, and RPL39L [63]. Lastly, we tested non-seed module genes whose closest seed neighbor was the ERAD-associated protein SYVN1, as our previous studies demonstrated that SYVN1 knockdown restored partial function to ΔF508-CFTR [19]. ...
... Additional enriched terms included cotranslational protein targeting to membrane, protein targeting to ER, translational elongation, ribosomal large subunit biogenesis, translational initiation, and protein complex assembly. Lukacs and colleagues recently identified a component of the ribosomal stalk, RPL12, as an effector of ΔF508-CFTR [63]. Silencing of RPL12 slowed the rate of translation, while increasing the folding efficiency and conformational stability of ΔF508-CFTR. ...
Background
We previously reported that expression of a miR-138 mimic or knockdown of SIN3A in primary cultures of cystic fibrosis (CF) airway epithelia increased ΔF508-CFTR mRNA and protein levels, and partially restored CFTR-dependent chloride transport. Global mRNA transcript profiling in ΔF508-CFBE cells treated with miR-138 mimic or SIN3A siRNA identified two genes, SYVN1 and NEDD8 , whose inhibition significantly increased ΔF508-CFTR trafficking, maturation, and function. Little is known regarding the dynamic changes in the CFTR gene network during such rescue events. We hypothesized that analysis of condition-specific gene networks from transcriptomic data characterizing ΔF508-CFTR rescue could help identify dynamic gene modules associated with CFTR biogenesis.
Methods
We applied a computational method, termed M-module, to analyze multiple gene networks, each of which exhibited differential activity compared to a baseline condition. In doing so, we identified both unique and shared gene pathways across multiple differential networks. To construct differential networks, gene expression data from CFBE cells were divided into three groups: (1) siRNA inhibition of NEDD8 and SYVN1 ; (2) miR-138 mimic and SIN3A siRNA; and (3) temperature (27 °C for 24 h, 40 °C for 24 h, and 27 °C for 24 h followed by 40 °C for 24 h).
Results
Interrogation of individual networks (e.g., NEDD8/SYVN1 network), combinations of two networks (e.g., NEDD8/SYVN1 + temperature networks), and all three networks yielded sets of 1-modules, 2-modules, and 3-modules, respectively. Gene ontology analysis revealed significant enrichment of dynamic modules in pathways including translation, protein metabolic/catabolic processes, protein complex assembly, and endocytosis. Candidate CFTR effectors identified in the analysis included CHURC1, GZF1, and RPL15, and siRNA-mediated knockdown of these genes partially restored CFTR-dependent transepithelial chloride current to ΔF508-CFBE cells.
Conclusions
The ability of the M-module to identify dynamic modules involved in ΔF508 rescue provides a novel approach for studying CFTR biogenesis and identifying candidate suppressors of ΔF508.
