Gergely Lukacs | McGill University (original) (raw)
Papers by Gergely Lukacs
Developmental Cell, Mar 1, 2018
Highlights d RFFL accelerates lysosomal degradation of unfolded CFTR from the cell surface d RFFL... more Highlights d RFFL accelerates lysosomal degradation of unfolded CFTR from the cell surface d RFFL directly recognizes unfolded CFTR through the disordered regions d RFFL promotes K63-linked poly-ubiquitination of unfolded CFTR in post-Golgi d RFFL inhibition improves CFTR corrector efficacy
Frontiers in pharmacology, Apr 25, 2024
Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductan... more Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Premature termination codons (PTCs) represent~9% of CF mutations that typically cause severe expression defects of the CFTR anion channel. Despite the prevalence of PTCs as the underlying cause of genetic diseases, understanding the therapeutic susceptibilities of their molecular defects, both at the transcript and protein levels remains partially elucidated. Given that the molecular pathologies depend on the PTC positions in CF, multiple pharmacological interventions are required to suppress the accelerated nonsense-mediated mRNA decay (NMD), to correct the CFTR conformational defect caused by misincorporated amino acids, and to enhance the inefficient stop codon readthrough. The G418-induced readthrough outcome was previously investigated only in reporter models that mimic the impact of the local sequence context on PTC mutations in CFTR. To identify the misincorporated amino acids and their ratios for PTCs in the context of full-length CFTR readthrough, we developed an affinity purification (AP)-tandem mass spectrometry (AP-MS/MS) pipeline. We confirmed the incorporation of Cys, Arg, and Trp residues at the UGA stop codons of G542X, R1162X, and S1196X in CFTR. Notably, we observed that the Cys and Arg incorporation was favored over that of Trp into these CFTR PTCs, suggesting that the transcript sequence beyond the proximity of PTCs and/or other factors can impact the amino acid incorporation and full-length CFTR functional expression. Additionally, establishing the misincorporated amino acid ratios in the readthrough CFTR PTCs aided in maximizing the functional rescue efficiency of PTCs by optimizing CFTR modulator combinations. Collectively, our findings contribute to the
Journal of Cystic Fibrosis
Science Translational Medicine
Cyclic adenosine 3′,5′-monophosphate (cAMP)–elevating agents, such as β 2 -adrenergic receptor (β... more Cyclic adenosine 3′,5′-monophosphate (cAMP)–elevating agents, such as β 2 -adrenergic receptor (β 2 -AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by β 2 -ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asth...
PLOS Biology, 2016
<p><b>(A)</b> Box-whisker plot of the cell proliferation parameter L (time to r... more <p><b>(A)</b> Box-whisker plot of the cell proliferation parameter L (time to reach half-maximal carrying capacity) of the <i>yor1-Δ0</i> (gene deletion) strain (cyan) and the <i>yor1-ΔF670</i> mutant (magenta) following treatment with oligomycin at multiple concentrations. 287 cultures were analyzed for <i>yor1-Δ0</i> and 384 cultures for <i>yor1-ΔF670</i>. “N < 287” or “N < 384” indicates that the concentration of oligomycin was fully growth inhibitory for some of the replicate cultures. Box plots indicate the central 75% of values, whiskers the total value range and averages are indicated by black bars. At higher growth inhibitory concentrations, the phenotypic distributions widen, and thus the high range for the <i>yor1-Δ0</i> at 0.15 ug/mL oligomycin is not depicted to permit better visualization of the data. The single mutant reference strain data from panel A is dose-normalized and plotted in the background for the identically normalized double-mutant data (black dots) in panels B–E to illustrate how each gene deletion influences the oligomycin resistance associated with the <i>yor1-Δ0</i> and <i>yor1-ΔF</i> alleles. <b>(B)</b> The <i>RPL12A</i> deletion increases oligomycin resistance in the context of <i>yor1-ΔF670</i> (magenta), but not <i>yor1-Δ0</i> (cyan). Oligomycin resistance was compared between the single mutants (shown in panel A) and the respective double mutant cultures, separately constructed in quadruplicate (biological replicates, black circles). Data for each series of replicates was normalized by the difference in L in the absence of oligomcyin (orange circles). <b>(C–E)</b> Plots similar to those in panel B are shown for gene modules comprising the cytoplasmic exosome involved in mRNA regulation (C), genes functioning in the nuclear exosome and rRNA processing (D), and additional ribosomal proteins (E). See <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002462#pbio.1002462.t001" target="_blank">Table 1</a></b> for additional information. The underlying data of panels A–E can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002462#pbio.1002462.s001" target="_blank">S1 Data</a>.</p
Journal of Molecular Biology, 2016
Deletion of Phe508 in the nucleotide binding domain (∆F508-NBD1) of the cystic fibrosis transmemb... more Deletion of Phe508 in the nucleotide binding domain (∆F508-NBD1) of the cystic fibrosis transmembrane regulator (CFTR; a cyclic AMP-regulated chloride channel) is the most frequent mutation associated with cystic fibrosis. This mutation affects the maturation and gating of CFTR protein. The search for new high-affinity ligands of CFTR acting as dual modulators (correctors/activators) presents a major challenge in the pharmacology of cystic fibrosis. Snake venoms are a rich source of natural multifunctional proteins, potential binders of ion channels. In this study, we identified the CB subunit of crotoxin from Crotalus durissus terrificus as a new ligand and allosteric modulator of CFTR. We showed that CB interacts with NBD1 of both wild type and ∆F508CFTR and increases their chloride channel currents. The potentiating effect of CB on CFTR activity was demonstrated using electrophysiological techniques in Xenopuslaevis oocytes, in CFTR-HeLa cells, and ex vivo in mouse colon tissue. The correcting effect of CB was shown by functional rescue of CFTR activity after 24-h ΔF508CFTR treatments with CB. Moreover, the presence of fully glycosylated CFTR was observed. Molecular docking allowed us to propose a model of the complex involving of the ABCβ and F1-like ATP-binding subdomains of ΔF508-NBD1. Hydrogen-deuterium exchange analysis confirmed stabilization in these regions, also showing allosteric stabilization in two other distal regions. Surface plasmon resonance competition studies showed that CB disrupts the ∆F508CFTR-cytokeratin 8 complex, allowing for the escape of ∆F508CFTR from degradation. Therefore CB, as a dual modulator of ΔF508CFTR, constitutes a template for the development of new anti-CF agents.
bioRxiv (Cold Spring Harbor Laboratory), Oct 19, 2023
Scientific Reports, 2020
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel cause cystic ... more Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel cause cystic fibrosis. Chaperones, including HSC70, DNAJA1 and DNAJA2, play key roles in both the folding and degradation of wild-type and mutant CFTR at multiple cellular locations. DNAJA1 and HSC70 promote the folding of newly synthesized CFTR at the endoplasmic reticulum (ER), but are required for the rapid turnover of misfolded channel at the plasma membrane (PM). DNAJA2 and HSC70 are also involved in the ER-associated degradation (ERAD) of misfolded CFTR, while they assist the refolding of destabilized channel at the PM. These outcomes may depend on the binding of chaperones to specific sites within CFTR, which would be exposed in non-native states. A CFTR peptide library was used to identify binding sites for HSC70, DNAJA1 and DNAJA2, validated by competition and functional assays. Each chaperone had a distinct binding pattern, and sites were distributed between the surfaces of the CFTR cytosol...
Nature Structural & Molecular Biology, 2018
EMBO reports, 2018
Mutations in PINK1 cause autosomal recessive Parkinson's disease (PD), a neurodegenerative moveme... more Mutations in PINK1 cause autosomal recessive Parkinson's disease (PD), a neurodegenerative movement disorder. PINK1 is a kinase that acts as a sensor of mitochondrial damage and initiates Parkin-mediated clearance of the damaged organelle. PINK1 phosphorylates Ser65 in both ubiquitin and the ubiquitin-like (Ubl) domain of Parkin, which stimulates its E3 ligase activity. Autophosphorylation of PINK1 is required for Parkin activation, but how this modulates the ubiquitin kinase activity is unclear. Here, we show that autophosphorylation of Tribolium castaneum PINK1 is required for substrate recognition. Using enzyme kinetics and NMR spectroscopy, we reveal that PINK1 binds the Parkin Ubl with a 10-fold higher affinity than ubiquitin via a conserved interface that is also implicated in RING1 and SH3 binding. The interaction requires phosphorylation at Ser205, an invariant PINK1 residue (Ser228 in human). Using mass spectrometry, we demonstrate that PINK1 rapidly autophosphorylates in trans at Ser205. Small-angle X-ray scattering and hydrogen-deuterium exchange experiments provide insights into the structure of the PINK1 catalytic domain. Our findings suggest that multiple PINK1 molecules autophosphorylate first prior to binding and phosphorylating ubiquitin and Parkin.
ABSTRACTCystic fibrosis (CF) is caused by mutations in a chloride channel called the human Cystic... more ABSTRACTCystic fibrosis (CF) is caused by mutations in a chloride channel called the human Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR). We used cryo-EM global conformational ensemble reconstruction to characterize the mechanism by which the breakthrough drug VX445 (Elexacaftor) simultaneously corrects both protein-folding and channel-gating defects caused by CF mutations. VX445 drives hCFTR molecules harboring the gating-defective G551D mutation towards the open-channel conformation by binding to a site in the first transmembrane domain. This binding interaction reverses the usual pathway of allosteric structural communication by which ATP binding activates channel conductance, which is blocked by the G551D mutation. Our ensemble reconstructions include a 3.4 Å non-native structure demonstrating that detachment of the first nucleotide-binding domain of hCFTR is directly coupled to local unfolding of the VX445 binding site. Reversal of this unfolding transition likely...
Journal of Cystic Fibrosis, 2019
Background: The potentiator ivacaftor (VX-770) has been approved for therapy of 38 cystic fibrosi... more Background: The potentiator ivacaftor (VX-770) has been approved for therapy of 38 cystic fibrosis (CF) mutations (∼10% of the patient population) associated with a gating defect of the CF transmembrane conductance regulator (CFTR). Despite the success of VX-770 treatment of patients carrying at least one allele of the most common gating mutation G551D-CFTR, some lung function decline and P. aeruginosa colonization persist. This study aims at identifying potentiator combinations that can considerably enhance the limited channel activity of a panel of CFTR gating mutants over monotherapy. Methods: The functional response of 13 CFTR mutants to single potentiators or systematic potentiator combinations was determined in the human bronchial epithelial cell line CFBE41o-and a subset of them was confirmed in primary human nasal epithelia (HNE). Results: In six out of thirteen CFTR missense mutants the fractional plasma membrane (PM) activity, a surrogate measure of CFTR channel gating, reached only ∼10-50% of WT channel activity upon VX-770 treatment, indicating incomplete gating correction. Combinatorial potentiator profiling and cluster analysis of mutant responses to 24 diverse investigational potentiators identified several compound pairs that improved the gating activity of R352Q-, S549R-, S549N-, G551D-, and G1244E-CFTR to ∼70-120% of the WT. Similarly, the potentiator combinations were able to confer WT-like function to G551D-CFTR in patient-derived human nasal epithelia. Conclusion: This study suggests that half of CF patients with missense mutations approved for VX-770 administration, could benefit from the development of dual potentiator therapy.
Scientific Reports, 2019
Deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulato... more Deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is the most frequent mutation causing cystic fibrosis (CF). F508del-CFTR is misfolded and prematurely degraded. Recently thymosin a-1 (Tα-1) was proposed as a single molecule-based therapy for CF, improving both F508del-CFTR maturation and function by restoring defective autophagy. However, three independent laboratories failed to reproduce these results. Lack of reproducibility has been ascribed by the authors of the original paper to the use of DMSO and to improper handling. Here, we address these potential issues by demonstrating that Tα-1 changes induced by DMSO are fully reversible and that Tα-1 peptides prepared from different stock solutions have equivalent biological activity. Considering the negative results here reported, six independent laboratories failed to demonstrate F508del-CFTR correction by Tα-1. This study also calls into question the autophagy m...
The Journal of general physiology, 1990
A novel, small conductance of Cl- channel was characterized by incorporation into planar bilayers... more A novel, small conductance of Cl- channel was characterized by incorporation into planar bilayers from a plasma membrane preparation of lobster walking leg nerves. Under conditions of symmetrical 100 mM NaCl, 10 mM Tris-HCl, pH 7.4, single Cl- channels exhibit rectifying current-voltage (I-V) behavior with a conductance of 19.2 +/- 0.8 pS at positive voltages and 15.1 +/- 1.6 pS in the voltage range of -40 to 0 mV. The channel exhibits a negligible permeability for Na+ compared with Cl- and displays the following sequence of anion permeability relative to Cl- as measured under near bi-ionic conditions: I- (2.7) greater than NO3- (1.8) greater than Br- (1.5) greater than Cl- (1.0) greater than CH3CO2- (0.18) greater than HCO3- (0.10) greater than gluconate (0.06) greater than F- (0.05). The unitary conductance saturates with increasing Cl- concentration in a Michaelis-Menten fashion with a Km of 100 mM and gamma max = 33 pS at positive voltage. The I-V curve is similar in 10 mM Tris ...
PLOS Biology, 2016
<p><b>(A, B)</b><i>RPL12</i> ablation increases the conformational ... more <p><b>(A, B)</b><i>RPL12</i> ablation increases the conformational stability of the solubilised rΔF508-CFTR. The thermoaggregation propensity of rΔF508-CFTR as a surrogate indicator of the channel conformational stability was determined in cell lysates of HeLa cells transfected with <i>RPL12</i> or NT siRNA in comparison to WT-CFTR. To minimize the amount of core-glycosylated form (filled arrowhead), cells were treated with CHX (2 h, 100 μg/ml) prior to lysis. Cell lysates were incubated for 15 min at 20°C–80°C followed by the sedimentation of aggregates and visualizing the remaining soluble channels by immunoblotting (A). The complex-glycosylated channel (empty arrowhead) was quantified by densitometry (B, <i>n</i> = 3–5). <b>(C)</b> Stability of ΔF508-CFTR after (left panel) or without (right panel) low-temperature rescue in HeLa cells upon <i>RPL12</i> knockdown was determined by immunoblot with CHX chase. <b>(D, E)</b> The complex-glycosylated (D, open arrowhead in C) or core-glycosylated CFTR (E, filled arrowhead in C) disappearance was quantified by densitometry and is expressed as percent of the initial amount (<i>n</i> = 3). <b>(F, G)</b> The effect of <i>RPL12</i> silencing on the PM stability (F, <i>n</i> = 4) and functional stability (G, n = 3) of rΔF508-CFTR after 1.5 and 3 h chase at 37°C. Same values as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002462#pbio.1002462.g002" target="_blank">Fig 2E and 2F</a> depicted as chase time-dependent percent remaining. <b>(G)</b> The effect of <i>RPL12</i> knockdown on the stability of metabolically labeled core-glycosylated ΔF508-CFTR in CFBE. Labeling was performed for 3 h at 26°C followed by chase for 2 h at 37°C. *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001. Error bars show SEM of 3–5 independent experiments. The underlying data of panels B and D–H can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002462#pbio.1002462.s001" target="_blank">S1 Data</a>.</p
Journal of Biological Chemistry, 1990
Scientific Reports, 2019
Impaired functional plasma membrane (PM) expression of the hERG K+-channel is associated with Lon... more Impaired functional plasma membrane (PM) expression of the hERG K+-channel is associated with Long-QT syndrome type-2 (LQT2) and increased risk of cardiac arrhythmia. Reduced PM-expression is primarily attributed to retention and degradation of misfolded channels by endoplasmic reticulum (ER) protein quality control (QC) systems. However, as the molecular pathogenesis of LQT2 was defined using severely-misfolded hERG variants with limited PM-expression, the potential contribution of post-ER (peripheral) QC pathways to the disease phenotype remains poorly established. Here, we investigate the cellular processing of mildly-misfolded Per-Arnt-Sim (PAS)-domain mutant hERGs, which display incomplete ER-retention and PM-expression defects at physiological temperature. We show that the attenuated PM-expression of hERG is dictated by mutation-specific contributions from both the ER and peripheral QC systems. At the ER, PAS-mutants experience inefficient conformational maturation coupled wit...
The Journal of biological chemistry, Dec 23, 2017
PD-L1 (programmed death ligand 1) and PD-L2 are cell surface glycoproteins that interact with pro... more PD-L1 (programmed death ligand 1) and PD-L2 are cell surface glycoproteins that interact with programmed death 1 (PD-1) on T cells to attenuate inflammation. PD-1 signaling has attracted intense interest for its role in a pathophysiological context: suppression of anti-tumor immunity. Similarly, vitamin D signaling has been increasingly investigated for its non-classical actions in stimulation of innate immunity and suppression of inflammatory responses. Here, we show that hormonal 1,25-dihydroxyvitamin D (1,25D) is a direct transcriptional inducer of the human genes encoding PD-L1 and PD-L2 through the vitamin D receptor (VDR), a ligand-regulated transcription factor. 1,25D stimulated transcription of the gene encoding PD-L1 in epithelial and myeloid cells, whereas the gene encoding the more tissue-restricted PD-L2 was regulated only in myeloid cells. We identified and characterized vitamin D response elements (VDREs) located in both genes and showed that 1,25D treatment induces ce...
Developmental Cell, Mar 1, 2018
Highlights d RFFL accelerates lysosomal degradation of unfolded CFTR from the cell surface d RFFL... more Highlights d RFFL accelerates lysosomal degradation of unfolded CFTR from the cell surface d RFFL directly recognizes unfolded CFTR through the disordered regions d RFFL promotes K63-linked poly-ubiquitination of unfolded CFTR in post-Golgi d RFFL inhibition improves CFTR corrector efficacy
Frontiers in pharmacology, Apr 25, 2024
Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductan... more Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Premature termination codons (PTCs) represent~9% of CF mutations that typically cause severe expression defects of the CFTR anion channel. Despite the prevalence of PTCs as the underlying cause of genetic diseases, understanding the therapeutic susceptibilities of their molecular defects, both at the transcript and protein levels remains partially elucidated. Given that the molecular pathologies depend on the PTC positions in CF, multiple pharmacological interventions are required to suppress the accelerated nonsense-mediated mRNA decay (NMD), to correct the CFTR conformational defect caused by misincorporated amino acids, and to enhance the inefficient stop codon readthrough. The G418-induced readthrough outcome was previously investigated only in reporter models that mimic the impact of the local sequence context on PTC mutations in CFTR. To identify the misincorporated amino acids and their ratios for PTCs in the context of full-length CFTR readthrough, we developed an affinity purification (AP)-tandem mass spectrometry (AP-MS/MS) pipeline. We confirmed the incorporation of Cys, Arg, and Trp residues at the UGA stop codons of G542X, R1162X, and S1196X in CFTR. Notably, we observed that the Cys and Arg incorporation was favored over that of Trp into these CFTR PTCs, suggesting that the transcript sequence beyond the proximity of PTCs and/or other factors can impact the amino acid incorporation and full-length CFTR functional expression. Additionally, establishing the misincorporated amino acid ratios in the readthrough CFTR PTCs aided in maximizing the functional rescue efficiency of PTCs by optimizing CFTR modulator combinations. Collectively, our findings contribute to the
Journal of Cystic Fibrosis
Science Translational Medicine
Cyclic adenosine 3′,5′-monophosphate (cAMP)–elevating agents, such as β 2 -adrenergic receptor (β... more Cyclic adenosine 3′,5′-monophosphate (cAMP)–elevating agents, such as β 2 -adrenergic receptor (β 2 -AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by β 2 -ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asth...
PLOS Biology, 2016
<p><b>(A)</b> Box-whisker plot of the cell proliferation parameter L (time to r... more <p><b>(A)</b> Box-whisker plot of the cell proliferation parameter L (time to reach half-maximal carrying capacity) of the <i>yor1-Δ0</i> (gene deletion) strain (cyan) and the <i>yor1-ΔF670</i> mutant (magenta) following treatment with oligomycin at multiple concentrations. 287 cultures were analyzed for <i>yor1-Δ0</i> and 384 cultures for <i>yor1-ΔF670</i>. “N < 287” or “N < 384” indicates that the concentration of oligomycin was fully growth inhibitory for some of the replicate cultures. Box plots indicate the central 75% of values, whiskers the total value range and averages are indicated by black bars. At higher growth inhibitory concentrations, the phenotypic distributions widen, and thus the high range for the <i>yor1-Δ0</i> at 0.15 ug/mL oligomycin is not depicted to permit better visualization of the data. The single mutant reference strain data from panel A is dose-normalized and plotted in the background for the identically normalized double-mutant data (black dots) in panels B–E to illustrate how each gene deletion influences the oligomycin resistance associated with the <i>yor1-Δ0</i> and <i>yor1-ΔF</i> alleles. <b>(B)</b> The <i>RPL12A</i> deletion increases oligomycin resistance in the context of <i>yor1-ΔF670</i> (magenta), but not <i>yor1-Δ0</i> (cyan). Oligomycin resistance was compared between the single mutants (shown in panel A) and the respective double mutant cultures, separately constructed in quadruplicate (biological replicates, black circles). Data for each series of replicates was normalized by the difference in L in the absence of oligomcyin (orange circles). <b>(C–E)</b> Plots similar to those in panel B are shown for gene modules comprising the cytoplasmic exosome involved in mRNA regulation (C), genes functioning in the nuclear exosome and rRNA processing (D), and additional ribosomal proteins (E). See <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002462#pbio.1002462.t001" target="_blank">Table 1</a></b> for additional information. The underlying data of panels A–E can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002462#pbio.1002462.s001" target="_blank">S1 Data</a>.</p
Journal of Molecular Biology, 2016
Deletion of Phe508 in the nucleotide binding domain (∆F508-NBD1) of the cystic fibrosis transmemb... more Deletion of Phe508 in the nucleotide binding domain (∆F508-NBD1) of the cystic fibrosis transmembrane regulator (CFTR; a cyclic AMP-regulated chloride channel) is the most frequent mutation associated with cystic fibrosis. This mutation affects the maturation and gating of CFTR protein. The search for new high-affinity ligands of CFTR acting as dual modulators (correctors/activators) presents a major challenge in the pharmacology of cystic fibrosis. Snake venoms are a rich source of natural multifunctional proteins, potential binders of ion channels. In this study, we identified the CB subunit of crotoxin from Crotalus durissus terrificus as a new ligand and allosteric modulator of CFTR. We showed that CB interacts with NBD1 of both wild type and ∆F508CFTR and increases their chloride channel currents. The potentiating effect of CB on CFTR activity was demonstrated using electrophysiological techniques in Xenopuslaevis oocytes, in CFTR-HeLa cells, and ex vivo in mouse colon tissue. The correcting effect of CB was shown by functional rescue of CFTR activity after 24-h ΔF508CFTR treatments with CB. Moreover, the presence of fully glycosylated CFTR was observed. Molecular docking allowed us to propose a model of the complex involving of the ABCβ and F1-like ATP-binding subdomains of ΔF508-NBD1. Hydrogen-deuterium exchange analysis confirmed stabilization in these regions, also showing allosteric stabilization in two other distal regions. Surface plasmon resonance competition studies showed that CB disrupts the ∆F508CFTR-cytokeratin 8 complex, allowing for the escape of ∆F508CFTR from degradation. Therefore CB, as a dual modulator of ΔF508CFTR, constitutes a template for the development of new anti-CF agents.
bioRxiv (Cold Spring Harbor Laboratory), Oct 19, 2023
Scientific Reports, 2020
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel cause cystic ... more Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel cause cystic fibrosis. Chaperones, including HSC70, DNAJA1 and DNAJA2, play key roles in both the folding and degradation of wild-type and mutant CFTR at multiple cellular locations. DNAJA1 and HSC70 promote the folding of newly synthesized CFTR at the endoplasmic reticulum (ER), but are required for the rapid turnover of misfolded channel at the plasma membrane (PM). DNAJA2 and HSC70 are also involved in the ER-associated degradation (ERAD) of misfolded CFTR, while they assist the refolding of destabilized channel at the PM. These outcomes may depend on the binding of chaperones to specific sites within CFTR, which would be exposed in non-native states. A CFTR peptide library was used to identify binding sites for HSC70, DNAJA1 and DNAJA2, validated by competition and functional assays. Each chaperone had a distinct binding pattern, and sites were distributed between the surfaces of the CFTR cytosol...
Nature Structural & Molecular Biology, 2018
EMBO reports, 2018
Mutations in PINK1 cause autosomal recessive Parkinson's disease (PD), a neurodegenerative moveme... more Mutations in PINK1 cause autosomal recessive Parkinson's disease (PD), a neurodegenerative movement disorder. PINK1 is a kinase that acts as a sensor of mitochondrial damage and initiates Parkin-mediated clearance of the damaged organelle. PINK1 phosphorylates Ser65 in both ubiquitin and the ubiquitin-like (Ubl) domain of Parkin, which stimulates its E3 ligase activity. Autophosphorylation of PINK1 is required for Parkin activation, but how this modulates the ubiquitin kinase activity is unclear. Here, we show that autophosphorylation of Tribolium castaneum PINK1 is required for substrate recognition. Using enzyme kinetics and NMR spectroscopy, we reveal that PINK1 binds the Parkin Ubl with a 10-fold higher affinity than ubiquitin via a conserved interface that is also implicated in RING1 and SH3 binding. The interaction requires phosphorylation at Ser205, an invariant PINK1 residue (Ser228 in human). Using mass spectrometry, we demonstrate that PINK1 rapidly autophosphorylates in trans at Ser205. Small-angle X-ray scattering and hydrogen-deuterium exchange experiments provide insights into the structure of the PINK1 catalytic domain. Our findings suggest that multiple PINK1 molecules autophosphorylate first prior to binding and phosphorylating ubiquitin and Parkin.
ABSTRACTCystic fibrosis (CF) is caused by mutations in a chloride channel called the human Cystic... more ABSTRACTCystic fibrosis (CF) is caused by mutations in a chloride channel called the human Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR). We used cryo-EM global conformational ensemble reconstruction to characterize the mechanism by which the breakthrough drug VX445 (Elexacaftor) simultaneously corrects both protein-folding and channel-gating defects caused by CF mutations. VX445 drives hCFTR molecules harboring the gating-defective G551D mutation towards the open-channel conformation by binding to a site in the first transmembrane domain. This binding interaction reverses the usual pathway of allosteric structural communication by which ATP binding activates channel conductance, which is blocked by the G551D mutation. Our ensemble reconstructions include a 3.4 Å non-native structure demonstrating that detachment of the first nucleotide-binding domain of hCFTR is directly coupled to local unfolding of the VX445 binding site. Reversal of this unfolding transition likely...
Journal of Cystic Fibrosis, 2019
Background: The potentiator ivacaftor (VX-770) has been approved for therapy of 38 cystic fibrosi... more Background: The potentiator ivacaftor (VX-770) has been approved for therapy of 38 cystic fibrosis (CF) mutations (∼10% of the patient population) associated with a gating defect of the CF transmembrane conductance regulator (CFTR). Despite the success of VX-770 treatment of patients carrying at least one allele of the most common gating mutation G551D-CFTR, some lung function decline and P. aeruginosa colonization persist. This study aims at identifying potentiator combinations that can considerably enhance the limited channel activity of a panel of CFTR gating mutants over monotherapy. Methods: The functional response of 13 CFTR mutants to single potentiators or systematic potentiator combinations was determined in the human bronchial epithelial cell line CFBE41o-and a subset of them was confirmed in primary human nasal epithelia (HNE). Results: In six out of thirteen CFTR missense mutants the fractional plasma membrane (PM) activity, a surrogate measure of CFTR channel gating, reached only ∼10-50% of WT channel activity upon VX-770 treatment, indicating incomplete gating correction. Combinatorial potentiator profiling and cluster analysis of mutant responses to 24 diverse investigational potentiators identified several compound pairs that improved the gating activity of R352Q-, S549R-, S549N-, G551D-, and G1244E-CFTR to ∼70-120% of the WT. Similarly, the potentiator combinations were able to confer WT-like function to G551D-CFTR in patient-derived human nasal epithelia. Conclusion: This study suggests that half of CF patients with missense mutations approved for VX-770 administration, could benefit from the development of dual potentiator therapy.
Scientific Reports, 2019
Deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulato... more Deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is the most frequent mutation causing cystic fibrosis (CF). F508del-CFTR is misfolded and prematurely degraded. Recently thymosin a-1 (Tα-1) was proposed as a single molecule-based therapy for CF, improving both F508del-CFTR maturation and function by restoring defective autophagy. However, three independent laboratories failed to reproduce these results. Lack of reproducibility has been ascribed by the authors of the original paper to the use of DMSO and to improper handling. Here, we address these potential issues by demonstrating that Tα-1 changes induced by DMSO are fully reversible and that Tα-1 peptides prepared from different stock solutions have equivalent biological activity. Considering the negative results here reported, six independent laboratories failed to demonstrate F508del-CFTR correction by Tα-1. This study also calls into question the autophagy m...
The Journal of general physiology, 1990
A novel, small conductance of Cl- channel was characterized by incorporation into planar bilayers... more A novel, small conductance of Cl- channel was characterized by incorporation into planar bilayers from a plasma membrane preparation of lobster walking leg nerves. Under conditions of symmetrical 100 mM NaCl, 10 mM Tris-HCl, pH 7.4, single Cl- channels exhibit rectifying current-voltage (I-V) behavior with a conductance of 19.2 +/- 0.8 pS at positive voltages and 15.1 +/- 1.6 pS in the voltage range of -40 to 0 mV. The channel exhibits a negligible permeability for Na+ compared with Cl- and displays the following sequence of anion permeability relative to Cl- as measured under near bi-ionic conditions: I- (2.7) greater than NO3- (1.8) greater than Br- (1.5) greater than Cl- (1.0) greater than CH3CO2- (0.18) greater than HCO3- (0.10) greater than gluconate (0.06) greater than F- (0.05). The unitary conductance saturates with increasing Cl- concentration in a Michaelis-Menten fashion with a Km of 100 mM and gamma max = 33 pS at positive voltage. The I-V curve is similar in 10 mM Tris ...
PLOS Biology, 2016
<p><b>(A, B)</b><i>RPL12</i> ablation increases the conformational ... more <p><b>(A, B)</b><i>RPL12</i> ablation increases the conformational stability of the solubilised rΔF508-CFTR. The thermoaggregation propensity of rΔF508-CFTR as a surrogate indicator of the channel conformational stability was determined in cell lysates of HeLa cells transfected with <i>RPL12</i> or NT siRNA in comparison to WT-CFTR. To minimize the amount of core-glycosylated form (filled arrowhead), cells were treated with CHX (2 h, 100 μg/ml) prior to lysis. Cell lysates were incubated for 15 min at 20°C–80°C followed by the sedimentation of aggregates and visualizing the remaining soluble channels by immunoblotting (A). The complex-glycosylated channel (empty arrowhead) was quantified by densitometry (B, <i>n</i> = 3–5). <b>(C)</b> Stability of ΔF508-CFTR after (left panel) or without (right panel) low-temperature rescue in HeLa cells upon <i>RPL12</i> knockdown was determined by immunoblot with CHX chase. <b>(D, E)</b> The complex-glycosylated (D, open arrowhead in C) or core-glycosylated CFTR (E, filled arrowhead in C) disappearance was quantified by densitometry and is expressed as percent of the initial amount (<i>n</i> = 3). <b>(F, G)</b> The effect of <i>RPL12</i> silencing on the PM stability (F, <i>n</i> = 4) and functional stability (G, n = 3) of rΔF508-CFTR after 1.5 and 3 h chase at 37°C. Same values as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002462#pbio.1002462.g002" target="_blank">Fig 2E and 2F</a> depicted as chase time-dependent percent remaining. <b>(G)</b> The effect of <i>RPL12</i> knockdown on the stability of metabolically labeled core-glycosylated ΔF508-CFTR in CFBE. Labeling was performed for 3 h at 26°C followed by chase for 2 h at 37°C. *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001. Error bars show SEM of 3–5 independent experiments. The underlying data of panels B and D–H can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002462#pbio.1002462.s001" target="_blank">S1 Data</a>.</p
Journal of Biological Chemistry, 1990
Scientific Reports, 2019
Impaired functional plasma membrane (PM) expression of the hERG K+-channel is associated with Lon... more Impaired functional plasma membrane (PM) expression of the hERG K+-channel is associated with Long-QT syndrome type-2 (LQT2) and increased risk of cardiac arrhythmia. Reduced PM-expression is primarily attributed to retention and degradation of misfolded channels by endoplasmic reticulum (ER) protein quality control (QC) systems. However, as the molecular pathogenesis of LQT2 was defined using severely-misfolded hERG variants with limited PM-expression, the potential contribution of post-ER (peripheral) QC pathways to the disease phenotype remains poorly established. Here, we investigate the cellular processing of mildly-misfolded Per-Arnt-Sim (PAS)-domain mutant hERGs, which display incomplete ER-retention and PM-expression defects at physiological temperature. We show that the attenuated PM-expression of hERG is dictated by mutation-specific contributions from both the ER and peripheral QC systems. At the ER, PAS-mutants experience inefficient conformational maturation coupled wit...
The Journal of biological chemistry, Dec 23, 2017
PD-L1 (programmed death ligand 1) and PD-L2 are cell surface glycoproteins that interact with pro... more PD-L1 (programmed death ligand 1) and PD-L2 are cell surface glycoproteins that interact with programmed death 1 (PD-1) on T cells to attenuate inflammation. PD-1 signaling has attracted intense interest for its role in a pathophysiological context: suppression of anti-tumor immunity. Similarly, vitamin D signaling has been increasingly investigated for its non-classical actions in stimulation of innate immunity and suppression of inflammatory responses. Here, we show that hormonal 1,25-dihydroxyvitamin D (1,25D) is a direct transcriptional inducer of the human genes encoding PD-L1 and PD-L2 through the vitamin D receptor (VDR), a ligand-regulated transcription factor. 1,25D stimulated transcription of the gene encoding PD-L1 in epithelial and myeloid cells, whereas the gene encoding the more tissue-restricted PD-L2 was regulated only in myeloid cells. We identified and characterized vitamin D response elements (VDREs) located in both genes and showed that 1,25D treatment induces ce...