Key points
Biochemical and biophysical characterizations of three nonsense mutations of cystic fibrosis transmembrane conductance regulator (CFTR) associated with a severe form of cystic fibrosis ...(CF) reveal the importance and heterogenous effects of the position of the premature termination codon (PTC) on the CFTR protein function.
Electrophysiological studies of W1282X‐CFTR, whose PTC is closer to the C‐terminus of CFTR, suggest the presence of both C‐terminus truncated CFTR proteins that are poorly functional and read‐through, full‐length products.
For G542X‐ and E60X‐CFTR, the only mechanism capable of generating functional proteins is the read‐through, but the outcome of read‐through products is highly variable depending on the interplay between the missense mutation caused by the read‐through and the structural context of the protein.
Pharmacological studies of these three PTCs with various CFTR modulators suggest position‐dependent therapeutic strategies for these disease‐inflicting mutations.
About one‐third of genetic diseases and cancers are caused by the introduction of premature termination codons (PTCs). In theory, the location of the PTC in a gene determines the alternative mechanisms of translation, including premature cessation or reinitiation of translation, and read‐through, resulting in differential effects on protein integrity. In this study, we used CFTR as a model system to investigate the positional effect of the PTC because of its well‐understood structure‐function relationship and pathophysiology. The characterization of three PTC mutations, E60X‐, G542X‐ and W1282X‐CFTR revealed heterogenous effects of these PTCs on CFTR function. The W1282X mutation results in both C‐terminus truncated and read‐through proteins that are partially or fully functional. In contrast, only the read‐through protein is functional with E60X‐ and G542X‐CFTR, although abundant N‐terminus truncated proteins due to reinitiation of translation were detected in E60X‐CFTR. Single‐channel studies of the read‐through proteins of E60X‐ and G542X‐CFTR demonstrated that both mutations have a single‐channel amplitude similar to wild type (WT), and good responses to high‐affinity ATP analogues, suggesting intact ion permeation pathways and nucleotide binding domains (NBDs), albeit with reduced open probability (Po). The comparison of the Po of these mutations with the proposed missense mutations revealed potential identities of the read‐through products. Importantly, a majority of the functional protein studied responds to CFTR modulators like GLPG1837 and Lumacaftor. These results not only expand current understanding of the molecular (patho)physiology of CFTR, but also infer therapeutic strategies for different PTC mutations at large.
Key points
Biochemical and biophysical characterizations of three nonsense mutations of cystic fibrosis transmembrane conductance regulator (CFTR) associated with a severe form of cystic fibrosis (CF) reveal the importance and heterogenous effects of the position of the premature termination codon (PTC) on the CFTR protein function.
Electrophysiological studies of W1282X‐CFTR, whose PTC is closer to the C‐terminus of CFTR, suggest the presence of both C‐terminus truncated CFTR proteins that are poorly functional and read‐through, full‐length products.
For G542X‐ and E60X‐CFTR, the only mechanism capable of generating functional proteins is the read‐through, but the outcome of read‐through products is highly variable depending on the interplay between the missense mutation caused by the read‐through and the structural context of the protein.
Pharmacological studies of these three PTCs with various CFTR modulators suggest position‐dependent therapeutic strategies for these disease‐inflicting mutations.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) chloride channel plays a critical role in regulating transepithelial movement of water and electrolyte in exocrine tissues. Malfunction ...of the channel because of mutations of the
gene results in CF, the most prevalent lethal genetic disease among Caucasians. Recently, the publication of atomic structures of CFTR in two distinct conformations provides, for the first time, a clear overview of the protein. However, given the highly dynamic nature of the interactions among CFTR's various domains, better understanding of the functional significance of these structures requires an integration of these new structural insights with previously established biochemical/biophysical studies, which is the goal of this review.
Key points
Electrophysiological characterization of Q1412X‐CFTR, a C‐terminal truncation mutation of cystic fibrosis transmembrane conductance regulator (CFTR) associated with the severe form of ...cystic fibrosis (CF), reveals a gating defect that has not been reported previously.
Mechanistic investigations of the gating deficit in Q1412X‐CFTR suggest that the reduced open probability in Q1412X‐CFTR is the result of a disruption of the function of the second ATP binding site (or site 2) in the nucleotide binding domains (NBDs).
Detailed comparisons of several mutations with different degrees of truncation in the C‐terminal region of NBD2 reveal the importance of the last two beta‐strands in NBD2 for maintaining proper gating functions.
The results of the present study also show that the application of clinically‐approved drugs (VX‐770 and VX‐809) can greatly enhance the function of Q1412X, providing in vitro evidence for a therapeutic strategy employing both reagents for patients bearing Q1412X or similar truncation mutations.
Cystic fibrosis (CF) is caused by loss‐of‐function mutations of cystic fibrosis transmembrane conductance regulator (CFTR), a phosphorylation‐activated but ATP‐gated chloride channel. Based on the molecular mechanism of CF pathogenesis, disease‐associated mutations are categorized into six classes. Among them, Class VI, whose members include some of the C‐terminal truncation mutations such as Q1412X, is defined as decreased membrane expression because of a faster turnover rate. In the present study, we characterized the functional properties of Q1412X‐CFTR, a severe‐form premature stop codon mutation. We confirmed previous findings of a ∼90% decrease in membrane expression but found a ∼95% reduction in the open probability (Po). Detailed kinetic studies support the idea that the gating defect is the result of a dysfunctional ATP‐binding site 2 in the nucleotide binding domains (NBDs). Because the Q1412X mutation results in a deletion of the last two beta‐strands in NBD2 and the whole C‐terminal region, we further characterized truncation mutations with different degrees of deletion in this segment. Mutations that completely or partially remove the C‐terminus of CFTR at the same time as keeping an intact NBD2 (i.e. D1425X and S1455X) assume gating function almost identical to that of wild‐type channels. However, the deletion of the last beta‐strand in the NBD2 (i.e. N1419X) causes gating dysfunction that is milder than that of Q1412X. Thus, normal CFTR gating requires structural integrity of NBD2. Moreover, our observation that clinically‐approved VX‐809 (Lumacaftor, Vertex Pharmaceuticals, Boston, MA, USA) and VX‐770 (Ivacaftor, Vertex Pharmaceuticals, Boston, MA, USA) significantly enhance the overall function of Q1412X‐CFTR provides the conceptual basis for the treatment of patients carrying this mutation.
Key points
Electrophysiological characterization of Q1412X‐CFTR, a C‐terminal truncation mutation of cystic fibrosis transmembrane conductance regulator (CFTR) associated with the severe form of cystic fibrosis (CF), reveals a gating defect that has not been reported previously.
Mechanistic investigations of the gating deficit in Q1412X‐CFTR suggest that the reduced open probability in Q1412X‐CFTR is the result of a disruption of the function of the second ATP binding site (or site 2) in the nucleotide binding domains (NBDs).
Detailed comparisons of several mutations with different degrees of truncation in the C‐terminal region of NBD2 reveal the importance of the last two beta‐strands in NBD2 for maintaining proper gating functions.
The results of the present study also show that the application of clinically‐approved drugs (VX‐770 and VX‐809) can greatly enhance the function of Q1412X, providing in vitro evidence for a therapeutic strategy employing both reagents for patients bearing Q1412X or similar truncation mutations.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Modulation of CFTR gating by permeant ions Yeh, Han-I; Yeh, Jiunn-Tyng; Hwang, Tzyh-Chang
The Journal of general physiology,
01/2015, Volume:
145, Issue:
1
Journal Article
Peer reviewed
Open access
Cystic fibrosis transmembrane conductance regulator (CFTR) is unique among ion channels in that after its phosphorylation by protein kinase A (PKA), its ATP-dependent gating violates microscopic ...reversibility caused by the intimate involvement of ATP hydrolysis in controlling channel closure. Recent studies suggest a gating model featuring an energetic coupling between opening and closing of the gate in CFTR's transmembrane domains and association and dissociation of its two nucleotide-binding domains (NBDs). We found that permeant ions such as nitrate can increase the open probability (Po) of wild-type (WT) CFTR by increasing the opening rate and decreasing the closing rate. Nearly identical effects were seen with a construct in which activity does not require phosphorylation of the regulatory domain, indicating that nitrate primarily affects ATP-dependent gating steps rather than PKA-dependent phosphorylation. Surprisingly, the effects of nitrate on CFTR gating are remarkably similar to those of VX-770 (N-(2,4-Di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide), a potent CFTR potentiator used in clinics. These include effects on single-channel kinetics of WT CFTR, deceleration of the nonhydrolytic closing rate, and potentiation of the Po of the disease-associated mutant G551D. In addition, both VX-770 and nitrate increased the activity of a CFTR construct lacking NBD2 (ΔNBD2), indicating that these gating effects are independent of NBD dimerization. Nonetheless, whereas VX-770 is equally effective when applied from either side of the membrane, nitrate potentiates gating mainly from the cytoplasmic side, implicating a common mechanism for gating modulation mediated through two separate sites of action.
Cystic fibrosis transmembrane conductance regulator (CFTR), the culprit behind the genetic disease cystic fibrosis (CF), is a phosphorylation-activated, but ATP-gated anion channel. Studies of human ...CFTR over the past two decades have provided an in-depth understanding of how CFTR works as an ion channel despite its structural resemblance to ABC transporters. Recently-solved cryo-EM structures of unphosphorylated human and zebrafish CFTR (hCFTR and zCFTR), as well as phosphorylated ATP-bound zebrafish and human CFTR offer an unprecedented opportunity to understand CFTR's function at a molecular level. Interestingly, despite millions of years of phylogenetic distance between human and zebrafish, the structures of zCFTR and hCFTR exhibit remarkable similarities. In the current study, we characterized biophysical and pharmacological properties of zCFTR with the patch-clamp technique, and showed surprisingly very different functional properties between these two orthologs. First, while hCFTR has a single-channel conductance of 8.4 pS with a linear I-V curve, zCFTR shows an inwardly-rectified I-V relationship with a single-channel conductance of ~3.5 pS. Second, single-channel gating behaviors of phosphorylated zCFTR are very different from those of hCFTR, featuring a very low open probability Po (0.03 ± 0.02, vs. ~0.50 for hCFTR) with exceedingly long closed events and brief openings. In addition, unlike hCFTR where each open burst is clearly defined with rare short-lived flickery closures, the open bursts of zCFTR are not easily resolved. Third, although abolishing ATP hydrolysis by replacing the catalytic glutamate with glutamine (i.e., E1372Q) drastically prolongs the open bursts defined by the macroscopic relaxation analysis in zCFTR, the Po within a "locked-open" burst of E1372Q-zCFTR is only ~ 0.35 (vs. Po > 0.94 in E1371Q-hCFTR). Collectively, our data not only provide a reasonable explanation for the unexpected closed-state structure of phosphorylated E1372Q-zCFTR with a canonical ATP-bound dimer of the nucleotide binding domains (NBDs), but also implicate significant structural and functional differences between these two evolutionarily distant orthologs.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The variability of blood pressure (BPV) has been suggested as a clinical indicator for cognitive dysfunction, yet the results from clinical studies are variable. This study investigated the ...relationship between BPV and the risk of cognitive decline or dementia. Bibliographic databases, including PubMed, Scopus, and Embase, were searched systematically for longitudinal cohort studies with BPV measurements and neuropsychological examinations or dementia diagnosis. A traditional meta‐analysis with subgroup analysis, and a further dose‐response meta‐analysis were conducted. Twenty cohort studies with 7 924 168 persons were included in this review. The results showed that a higher systolic BPV (SBPV), when measured with the coefficient of variation (SBP‐CV) or standard deviation (SBP‐SD), was associated with a higher risk of all‐cause dementia diagnosis but not incidence of cognitive decline on neuropsychological examinations. In subgroup analysis, the effect was more prominent when using BPV of shorter timeframes, during shorter follow‐ups, or among the elderly aged more than 65 years. No dose‐response relationship could be found. Our study suggested possible positive associations between SBPV and the risk of dementia. Further studies are required to validate these findings.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Background
Renal dysfunction is common in patients with coronary artery disease. Due to the shared vascular pathogenesis between the two conditions, novel biomarkers such as the fatty acid‐binding ...protein‐3 (FABP‐3) have been proposed for diagnosis and prognosis prediction. This multicentre prospective cohort study investigates the association between FABP‐3 and renal dysfunction.
Hypothesis
We hypothesized that higher FABP‐3 levels are correlated to worse renal outcome.
Methods
Patients with chronic coronary syndrome were classified into three groups based on the initial serum FABP‐3 levels. The Chronic Kidney Disease Epidemiology Collaboration (CKD‐EPI) equation was used to estimate the patient's renal function. Renal events were defined as >25% and >50% decline in estimated glomerular filtration rate (eGFR). Cox multivariable regression was employed to delineate the correlation between FABP‐3 and renal dysfunction.
Results
A total of 1606 subjects were included. During a mean follow‐up of 35.9 months, there were 239 patients with eGFR >25% reduction and 60 patients with >50% reduction. In the Kaplan–Meier survival curve and log‐rank test, increased levels of FABP‐3 were significantly correlated with eGFR >25% reduction (p < .001) and >50% reduction (p < .001). Multivariate Cox regression model revealed that subjects with higher FABP‐3 exhibited a greater risk of eGFR >25% reduction (Group 2: hazard ratio HR = 2.328, 95% confidence interval CI = 1.521–3.562, p < .001; Group 3: HR = 3.054, 95% CI = 1.952–4.776, p < .001) and >50% reduction (Group 3: HR = 4.838, 95% CI = 1.722–13.591, p = .003).
Conclusions
Serum FABP‐3 may serve as a novel biomarker to predict eGFR decline in patients with chronic coronary syndrome.
For patients with chronic coronary syndrome, early detection and prevention of kidney injury are crucial. We found that a higher serum FABP‐3 level was correlated to an increased risk of estimated glomerular filtration rate (eGFR) decline. Serum FABP‐3 may serve as a novel biomarker to predict eGFR decline in these patients.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Cystic fibrosis (CF) is a genetic disease affects CFTR channel synthesis. While 90 percent of the CF patients now benefit from small molecule target therapies, this treatment has yet to extend to ...those bearing nonsense mutations. Studies of these rare mutations using cell lines with native pathological signatures of the disease may lead to breakthroughs in therapeutic development. Here, we report the generation of CF patient-derived induced pluripotent stem cells (iPSCs) carrying a nonsense mutation at position 308 (S308X). The pluripotency and genomic profile of the iPSC line was validated as a resource that can enable future research for CF.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
This systematic review assesses the likelihood of developing dementia and cognitive impairment in patients with atrial fibrillation (AF) receiving non-vitamin K antagonist oral anticoagulants (NOACs) ...as opposed to vitamin K antagonists (VKAs).
We performed a systematic review with meta-analysis and trial sequential analysis (TSA), which encompassed both randomized controlled trials (RCTs) and observational studies. The objective was to assess the impact of NOACs and VKAs on the incidence of dementia in individuals diagnosed with AF.
Out of 1914 studies that were screened, 31 studies were included in the final analysis, which consisted of nine RCTs or their subsequent post-hoc analyses, in addition to 22 observational studies. The meta-analysis shows that NOACs were associated with a decreased probability of developing dementia of any cause Rate Ratio (RR): 0.88; 95 % confidence interval (95 % CI): 0.82–0.94, especially in patients below the age of 75 (RR: 0.78; 95 % CI: 0.73–0.84). Consistent patterns were observed across all forms of dementia and cognitive function decline. The overall evidence indicates notable variability in the outcome with a moderate-to-low degree of certainty. The TSA suggests that the total sample size of the included trials (155,647 patients) was significantly smaller than the required information size of 784,692 patients to discern the true effect of NOAC versus VKA in terms of reducing dementia risk.
NOACs may reduce the likelihood of developing dementia in patients with AF, particularly in those under the age of 75. This review highlights the urgent necessity for thorough research to determine the efficacy of NOACs in safeguarding cognitive health.
•The optimal treatment to lower incidence of dementia for AF patients remained elusive.•NOACs were associated with a decreased probability of developing all-cause dementia.•The negative association between NOACs and risk of dementia is more prominent in patients <75 years old.•Insufficient required information size was noted to determine the true effect of NOACs.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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