scholarly journals Different SUMO paralogues determine the fate of wild-type and mutant CFTRs: biogenesis versus degradation

2019 ◽  
Vol 30 (1) ◽  
pp. 4-16 ◽  
Author(s):  
Xiaoyan Gong ◽  
Yong Liao ◽  
Annette Ahner ◽  
Mads Breum Larsen ◽  
Xiaohui Wang ◽  
...  
Keyword(s):  
Physiological Role ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Specific Effects ◽  
Ubiquitin Conjugation ◽  
Protein Array Analysis ◽  
The Impact

A pathway for cystic fibrosis transmembrane conductance regulator (CFTR) degradation is initiated by Hsp27, which cooperates with Ubc9 and binds to the common F508del mutant to modify it with SUMO-2/3. These SUMO paralogues form polychains, which are recognized by the ubiquitin ligase, RNF4, for proteosomal degradation. Here, protein array analysis identified the SUMO E3, protein inhibitor of activated STAT 4 (PIAS4), which increased wild-type (WT) and F508del CFTR biogenesis in CFBE airway cells. PIAS4 increased immature CFTR threefold and doubled expression of mature CFTR, detected by biochemical and functional assays. In cycloheximide chase assays, PIAS4 slowed immature F508del degradation threefold and stabilized mature WT CFTR at the plasma membrance. PIAS4 knockdown reduced WT and F508del CFTR expression by 40–50%, suggesting a physiological role in CFTR biogenesis. PIAS4 modified F508del CFTR with SUMO-1 in vivo and reduced its conjugation to SUMO-2/3. These SUMO paralogue-specific effects of PIAS4 were reproduced in vitro using purified F508del nucleotide-binding domain 1 and SUMOylation reaction components. PIAS4 reduced endogenous ubiquitin conjugation to F508del CFTR by ∼50% and blocked the impact of RNF4 on mutant CFTR disposal. These findings indicate that different SUMO paralogues determine the fates of WT and mutant CFTRs, and they suggest that a paralogue switch during biogenesis can direct these proteins to different outcomes: biogenesis versus degradation.

scholarly journals Different SUMO Paralogs Determine the Fate of WT and Mutant CFTRs: Biogenesis vs. Degradation

2018 ◽  
Author(s):  
Xiaoyan Gong ◽  
Yong Liao ◽  
Annette Ahner ◽  
Mads Breum Larsen ◽  
Xiaohui Wang ◽  
...  
Keyword(s):  
Physiological Role ◽  
Array Analysis ◽  
Specific Effects ◽  
Ubiquitin Conjugation ◽  
Proteosomal Degradation ◽  
The Common ◽  
Protein Array Analysis ◽  
The Impact

ABSTRACTA pathway for CFTR degradation is initiated by Hsp27 which cooperates with Ubc9 and binds to the common F508del mutant to modify it with SUMO-2/3. These SUMO paralogs form poly-chains, which are recognized by the ubiquitin ligase, RNF4, for proteosomal degradation. Here, protein array analysis identified the SUMO E3, PIAS4, which increased WT and F508del CFTR biogenesis in CFBE airway cells. PIAS4 increased immature CFTR three-fold and doubled expression of mature CFTR, detected by biochemical and functional assays. In cycloheximide chase assays, PIAS4 slowed immature F508del degradation 3-fold and stabilized mature WT CFTR at the PM. PIAS4 knockdown reduced WT and F508del CFTR expression by 40-50%, suggesting a physiological role in CFTR biogenesis. PIAS4 modified F508del CFTR with SUMO-1in vivoand reduced its conjugation to SUMO-2/3. These SUMO paralog specific effects of PIAS4 were reproducedin vitrousing purified F508del NBD1 and SUMOylation reaction components. PIAS4 reduced endogenous ubiquitin conjugation to F508del CFTR by ~50%, and blocked the impact of RNF4 on mutant CFTR disposal. These findings indicate that different SUMO paralogs determine the fates of WT and mutant CFTRs, and they suggest that a paralog switch during biogenesis can direct these proteins to different outcomes: biogenesis vs. degradation.

scholarly journals Production of Noncapped Genomic RNAs Is Critical to Sindbis Virus Disease and Pathogenicity

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Autumn T. LaPointe ◽  
V Douglas Landers ◽  
Claire E. Westcott ◽  
Kevin J. Sokoloski
Keyword(s):  
Sindbis Virus ◽  
Virus Disease ◽  
Severe Disease ◽  
Wild Type Virus ◽  
Wild Type ◽  
Genomic Rnas ◽  
Mortality And Morbidity ◽  
The Impact

ABSTRACT Alphaviruses are positive-sense RNA viruses that utilize a 5′ cap structure to facilitate translation of viral proteins and to protect the viral RNA genome. Nonetheless, significant quantities of viral genomic RNAs that lack a canonical 5′ cap structure are produced during alphaviral replication and packaged into viral particles. However, the role/impact of the noncapped genomic RNA (ncgRNA) during alphaviral infection in vivo has yet to be characterized. To determine the importance of the ncgRNA in vivo, the previously described D355A and N376A nsP1 mutations, which increase or decrease nsP1 capping activity, respectively, were incorporated into the neurovirulent AR86 strain of Sindbis virus to enable characterization of the impact of altered capping efficiency in a murine model of infection. Mice infected with the N376A nsP1 mutant exhibited slightly decreased rates of mortality and delayed weight loss and neurological symptoms, although levels of inflammation in the brain were similar to those of wild-type infection. Although the D355A mutation resulted in decreased antiviral gene expression and increased resistance to interferon in vitro, mice infected with the D355A mutant showed significantly reduced mortality and morbidity compared to mice infected with wild-type virus. Interestingly, expression of proinflammatory cytokines was found to be significantly decreased in mice infected with the D355A mutant, suggesting that capping efficiency and the production of ncgRNA are vital to eliciting pathogenic levels of inflammation. Collectively, these data indicate that the ncgRNA have important roles during alphaviral infection and suggest a novel mechanism by which noncapped viral RNAs aid in viral pathogenesis. IMPORTANCE Mosquito-transmitted alphaviruses have been the cause of widespread outbreaks of disease that can range from mild illness to lethal encephalitis or severe polyarthritis. There are currently no safe and effective vaccines or therapeutics with which to prevent or treat alphaviral disease, highlighting the need to better understand alphaviral pathogenesis to develop novel antiviral strategies. This report reveals production of noncapped genomic RNAs (ncgRNAs) to be a novel determinant of alphaviral virulence and offers insight into the importance of inflammation to pathogenesis. Taken together, the findings reported here suggest that the ncgRNAs contribute to alphaviral pathogenesis through the sensing of the ncgRNAs during alphaviral infection and are necessary for the development of severe disease.

In vivo activation of CFTR-dependent chloride transport in murine airway epithelium by CNP

1997 ◽  
Vol 273 (5) ◽  
pp. L1065-L1072 ◽  
Author(s):  
Thomas J. Kelley ◽  
Calvin U. Cotton ◽  
Mitchell L. Drumm
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Transport ◽  
Nasal Epithelium ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Cyclic Monophosphate ◽  
Δf508 Cftr ◽  
Membrane Bound

Inhibitors of guanosine 3′,5′-cyclic monophosphate (cGMP)-inhibited phosphodiesterases stimulate Cl− transport across the nasal epithelia of cystic fibrosis mice carrying the ΔF508 mutation [cystic fibrosis transmembrane conductance regulator (CFTR) (ΔF/ΔF)], suggesting a role for cGMP in regulation of epithelial ion transport. Here we show that activation of membrane-bound guanylate cyclases by C-type natriuretic peptide (CNP) stimulates hyperpolarization of nasal epithelium in both wild-type and ΔF508 CFTR mice in vivo but not in nasal epithelium of mice lacking CFTR [CFTR(−/−)]. With the use of a nasal transepithelial potential difference (TEPD) assay, CNP was found to hyperpolarize lumen negative TEPD by 6.1 ± 0.6 mV in mice carrying wild-type CFTR. This value is consistent with that obtained with 8-bromoguanosine 3′,5′-cyclic monophosphate (6.2 ± 0.9 mV). A combination of the adenylate cyclase agonist forskolin and CNP demonstrated a synergistic ability to induce Cl− secretion across the nasal epithelium of CFTR(ΔF/ΔF) mice. No effect on TEPD was seen with this combination when used on CFTR(−/−) mice, implying that the CNP-induced change in TEPD in CFTR(ΔF/ΔF) mice is CFTR dependent.

Contribution of R domain phosphoserines to the function of CFTR studied in Fischer rat thyroid epithelia

2000 ◽  
Vol 279 (5) ◽  
pp. L835-L841 ◽  
Author(s):  
Olafur Baldursson ◽  
Herbert A. Berger ◽  
Michael J. Welsh
Keyword(s):  
Functional Role ◽  
Transmembrane Conductance Regulator ◽  
Regulatory Domain ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Dependent Protein ◽  
Rat Thyroid ◽  
Cystic Fibrosis Transmembrane

The regulatory domain of cystic fibrosis transmembrane conductance regulator (CFTR) regulates channel activity when several serines are phosphorylated by cAMP-dependent protein kinase. To further define the functional role of individual phosphoserines, we studied CFTR containing previously studied and new serine to alanine mutations. We expressed these constructs in Fischer rat thyroid epithelia and measured transepithelial Cl− current. Mutation of four in vivo phosphorylation sites, Ser660, Ser737, Ser795, and Ser813 (S-Quad-A), substantially decreased cAMP-stimulated current, suggesting that these four sites account for most of the phosphorylation-dependent response. Mutation of either Ser660 or Ser813 alone significantly decreased current, indicating that these residues play a key role in phosphorylation-dependent stimulation. However, neither Ser660 nor Ser813 alone increased current to wild-type levels; both residues were required. Changing Ser737 to alanine increased current above wild-type levels, suggesting that phosphorylation of Ser737 may inhibit current in wild-type CFTR. These data help define the functional role of regulatory domain phosphoserines and suggest interactions between individual phosphoserines.

A2 adenosine receptors regulate CFTR through PKA and PLA2

2002 ◽  
Vol 282 (1) ◽  
pp. L12-L25 ◽  
Author(s):  
B. R. Cobb ◽  
F. Ruiz ◽  
C. M. King ◽  
J. Fortenberry ◽  
H. Greer ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Receptor Activation ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Δf508 Cftr ◽  
Kinase A

We investigated adenosine (Ado) activation of the cystic fibrosis transmembrane conductance regulator (CFTR) in vitro and in vivo. A2B Ado receptors were identified in Calu-3, IB-3-1, COS-7, and primary human airway cells. Ado elevated cAMP in Calu-3, IB-3-1, and COS-7 cells and activated protein kinase A-dependent halide efflux in Calu-3 cells. Ado promoted arachidonic acid release from Calu-3 cells, and phospholipase A2(PLA2) inhibition blocked Ado-activated halide efflux in Calu-3 and COS-7 cells expressing CFTR. Forskolin- and β2-adrenergic receptor-stimulated efflux were not affected by the same treatment. Cytoplasmic PLA2(cPLA2) was identified in Calu-3, IB-3-1, and COS-7 cells, but cPLA2 inhibition did not affect Ado-stimulated cAMP concentrations. In cftr(+) and cftr(−/−) mice, Ado stimulated nasal Cl− secretion that was CFTR dependent and sensitive to A2 receptor and PLA2 blockade. In COS-7 cells transiently expressing ΔF508 CFTR, Ado activated halide efflux. Ado also activated G551D CFTR-dependent halide efflux when combined with arachidonic acid and phosphodiesterase inhibition. In conclusion, PLA2 and protein kinase A both contribute to A2 receptor activation of CFTR, and components of this signaling pathway can augment wild-type and mutant CFTR activity.

scholarly journals Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein

1999 ◽  
Vol 277 (4) ◽  
pp. C833-C839 ◽  
Author(s):  
Beate Illek ◽  
Lei Zhang ◽  
Nancy C. Lewis ◽  
Richard B. Moss ◽  
Jian-Yun Dong ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Adrenergic Stimulation ◽  
Transmembrane Conductance ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Nasal Potential Difference ◽  
Cystic Fibrosis Transmembrane

The patch-clamp technique was used to investigate the effects of the isoflavone genistein on disease-causing mutations (G551D and ΔF508) of the cystic fibrosis transmembrane conductance regulator (CFTR). In HeLa cells recombinantly expressing the trafficking-competent G551D-CFTR, the forskolin-stimulated Cl currents were small, and average open probability of G551D-CFTR was P o = 0.047 ± 0.019. Addition of genistein activated Cl currents ∼10-fold, and the P o of G551D-CFTR increased to 0.49 ± 0.12, which is a P o similar to wild-type CFTR. In cystic fibrosis (CF) epithelial cells homozygous for the trafficking-impaired ΔF508 mutation, forskolin and genistein activated Cl currents only after 4-phenylbutyrate treatment. These data suggested that genistein activated CFTR mutants that were present in the cell membrane. Therefore, we tested the effects of genistein in CF patients with the G551D mutation in nasal potential difference (PD) measurements in vivo. The perfusion of the nasal mucosa of G551D CF patients with isoproterenol had no effect; however, genistein stimulated Cl-dependent nasal PD by, on average, −2.4 ± 0.6 mV, which corresponds to 16.9% of the responses (to β-adrenergic stimulation) found in healthy subjects.

scholarly journals The human DnaJ homologue (Hdj)-1/heat-shock protein (Hsp) 40 co-chaperone is required for the in vivo stabilization of the cystic fibrosis transmembrane conductance regulator by Hsp70

2002 ◽  
Vol 366 (3) ◽  
pp. 797-806 ◽  
Author(s):  
Carlos M. FARINHA ◽  
Paulo NOGUEIRA ◽  
Filipa MENDES ◽  
Deborah PENQUE ◽  
Margarida D. AMARAL
Keyword(s):  
Cystic Fibrosis ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Hsc 70 ◽  
Immature Form ◽  
Cystic Fibrosis Transmembrane

The CFTR (cystic fibrosis transmembrane conductance regulator) gene, defective in cystic fibrosis, codes for a polytopic apical membrane protein functioning as a chloride channel. Wild-type (wt) CFTR matures inefficiently and CFTR with a deletion of Phe-508 (F508del), the most frequent mutation, is substantially retained as a core-glycosylated intermediate in the endoplasmic reticulum (ER), probably due to misfolding that is recognized by the cellular quality control machinery involving molecular chaperones. Here, we overexpressed the heat-shock protein (Hsp) 70 chaperone in vivo and observed no changes in degradation rate of the core-glycosylated form, nor in the efficiency of its conversion into the fully glycosylated form, for either wt- or F508del-CFTR, contrary to previous in vitro studies on the affect of heat-shock cognate (Hsc) 70 on part of the first nucleotide-binding domain of CFTR. Co-transfection of Hsp70 with its co-chaperone human DnaJ homologue (Hdj)-1/Hsp40, however, stabilizes the immature form of wt-CFTR, but not of F508del-CFTR, suggesting that these chaperones act on a wt-specific conformation. As the efficiency of conversion into the fully glycosylated form is not increased under Hsp70/Hdj-1 overexpression, the lack of these two chaperones does not seem to be critical for CFTR maturation and ER retention. The effects of 4-phenylbutyrate and deoxyspergualin, described previously to interfere with Hsp70 binding, were also tested upon CFTR degradation and processing. The sole effect observed was destabilization of F508del-CFTR.

scholarly journals The experimentally obtained functional impact assessments of GT>GC 5’ splice site variants differ markedly from those predicted

2019 ◽  
Author(s):  
Jian-Min Chen ◽  
Jin-Huan Lin ◽  
Emmanuelle Masson ◽  
Zhuan Liao ◽  
Claude Férec ◽  
...  
Keyword(s):  
Splice Site ◽  
Disease Genes ◽  
Wild Type ◽  
Human Genetic Disease ◽  
Functional Impact ◽  
Prediction Tools ◽  
Human Disease Genes ◽  
The Impact

ABSTRACTGT>GC 5’ splice site (or +2T>C) variants have been frequently reported to cause human genetic disease. However, although we have demonstrated that GT>GC variants in human disease genes may not invariably be pathogenic, none of the currently available splicing prediction tools appear to be capable of reliably distinguishing those GT>GC variants that generate wild-type transcripts from those that do not. Recently, SpliceAI, a novel deep residual neural network tool, has been developed for splicing prediction. Methodologically distinct from previous approaches that either rely on human-engineered features and/or which focus on short nucleotide windows adjoining exon-intron boundaries, SpliceAI assesses splicing determinants by evaluating 10,000 nucleotides of flanking contextual sequence to predict the functional role in splicing of each position in the pre-mRNA transcript. Herein, we evaluated the performance of SpliceAI in the context of three datasets of GT>GC variants, all of which had been characterized functionally in terms of their impact on mRNA splicing. The first two datasets refer to our recently described “in vivo” dataset of 45 disease-causing GT>GC variants and the “in vitro” dataset of 103 GT>GC substitutions. The third dataset comprised 12 BRCA1 GT>GC variants that were recently analyzed by saturation genome editing. We processed all GT>GC variants using the default settings of SpliceAI. Comparison of the SpliceAI-predicted and experimentally obtained functional impact assessments of the analyzed GT>GC variants revealed that although SpliceAI performed rather better than other prediction tools, it was still far from perfect. A key issue is that the impact of GT>GC (as well as GT>GA or +2T>A) variants that generated wild-type transcripts represents a quantitative change that can vary from barely detectable to almost full expression of wild-type transcripts, with wild-type transcripts often co-existing with aberrantly spliced transcripts. Our findings highlight the challenges that we still face in attempting to accurately identify splice-altering variants.

scholarly journals Characterization of genetically modified mice for phosphoglycerate mutase, a vitally-essential enzyme in glycolysis

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250856
Author(s):  
Takumi Mikawa ◽  
Eri Shibata ◽  
Midori Shimada ◽  
Ken Ito ◽  
Tomiko Ito ◽  
...  
Keyword(s):  
Glucose Metabolism ◽  
Knockout Mice ◽  
Physiological Role ◽  
Phosphoglycerate Mutase ◽  
Physiological Homeostasis ◽  
Essential Enzyme ◽  
The Impact

Glycolytic metabolism is closely involved in physiological homeostasis and pathophysiological states. Among glycolytic enzymes, phosphoglycerate mutase (PGAM) has been reported to exert certain physiological role in vitro, whereas its impact on glucose metabolism in vivo remains unclear. Here, we report the characterization of Pgam1 knockout mice. We observed that homozygous knockout mice of Pgam1 were embryonic lethal. Although we previously reported that both PGAM-1 and -2 affect global glycolytic profile of cancers in vitro, in vivo glucose parameters were less affected both in the heterozygous knockout of Pgam1 and in Pgam2 transgenic mice. Thus, the impact of PGAM on in vivo glucose metabolism is rather complex than expected before.

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