scholarly journals New insights into structure and function of bis-phosphinic acid derivatives and implications for CFTR modulation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sara Bitam ◽  
Ahmad Elbahnsi ◽  
Geordie Creste ◽  
Iwona Pranke ◽  
Benoit Chevalier ◽  
...  
Keyword(s):  
Phosphinic Acid ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Transmembrane Conductance Regulator ◽  
Intracellular Loop ◽  
Respiratory Cells ◽  
Cftr Protein ◽  
Dynamics Simulations ◽  
And Function ◽  
Molecular Mode

AbstractC407 is a compound that corrects the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein carrying the p.Phe508del (F508del) mutation. We investigated the corrector effect of c407 and its derivatives on F508del-CFTR protein. Molecular docking and dynamics simulations combined with site-directed mutagenesis suggested that c407 stabilizes the F508del-Nucleotide Binding Domain 1 (NBD1) during the co-translational folding process by occupying the position of the p.Phe1068 side chain located at the fourth intracellular loop (ICL4). After CFTR domains assembly, c407 occupies the position of the missing p.Phe508 side chain. C407 alone or in combination with the F508del-CFTR corrector VX-809, increased CFTR activity in cell lines but not in primary respiratory cells carrying the F508del mutation. A structure-based approach resulted in the synthesis of an extended c407 analog G1, designed to improve the interaction with ICL4. G1 significantly increased CFTR activity and response to VX-809 in primary nasal cells of F508del homozygous patients. Our data demonstrate that in-silico optimized c407 derivative G1 acts by a mechanism different from the reference VX-809 corrector and provide insights into its possible molecular mode of action. These results pave the way for novel strategies aiming to optimize the flawed ICL4–NBD1 interface.

scholarly journals Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Ubiquitylation as a Novel Pharmaceutical Target for Cystic Fibrosis

2020 ◽  
Vol 13 (4) ◽  
pp. 75 ◽  
Author(s):  
Ryosuke Fukuda ◽  
Tsukasa Okiyoneda
Keyword(s):  
Cystic Fibrosis ◽  
Structural Stability ◽  
Interacting Proteins ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Surrounding Environment ◽  
Cftr Protein ◽  
The Stability ◽  
And Function ◽  
Cystic Fibrosis Transmembrane

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene decrease the structural stability and function of the CFTR protein, resulting in cystic fibrosis. Recently, the effect of CFTR-targeting combination therapy has dramatically increased, and it is expected that add-on drugs that modulate the CFTR surrounding environment will further enhance their effectiveness. Various interacting proteins have been implicated in the structural stability of CFTR and, among them, molecules involved in CFTR ubiquitylation are promising therapeutic targets as regulators of CFTR degradation. This review focuses on the ubiquitylation mechanism that contributes to the stability of mutant CFTR at the endoplasmic reticulum (ER) and post-ER compartments and discusses the possibility as a pharmacological target for cystic fibrosis (CF).

CFTR is functionally active in GnRH-expressing GT1–7 hypothalamic neurons

1999 ◽  
Vol 277 (3) ◽  
pp. C563-C571 ◽  
Author(s):  
Richard T. Weyler ◽  
Karin A. Yurko-Mauro ◽  
Ronald Rubenstein ◽  
Wouter J. W. Kollen ◽  
William Reenstra ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Neuronal Cell ◽  
Cftr Gene ◽  
Gene Transcript ◽  
Transmembrane Conductance Regulator ◽  
Hypothalamic Neurons ◽  
Cftr Protein ◽  
And Function ◽  
Human Brains ◽  
Gene Mrna

We have demonstrated the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, mRNA, and protein within the rat and human brains, in areas regulating sexual differentiation and function. We have found that GT1–7, a gonadotropin-releasing hormone (GnRH)-secreting hypothalamic neuronal cell line, expresses the CFTR gene, mRNA, and protein and cAMP-dependent 36Cl efflux. A linear 7-pS Cl− conductance, which is stimulated by ATP and cAMP analogs and inhibited by glibenclamide, consistent with CFTR activity, has been identified in GT1–7 cells. Antisense oligo(dN) generated against exon 10 of the CFTR gene transcript (mRNA) inhibit GnRH secretion into media [312 ± 73, 850 ± 150, 963 ± 304, and 912 ± 74 pg GnRH/4 × 106 cells for antisense, sense, missense, and no oligo(dN), respectively; P < 0.029 for antisense oligo(dN)-treated vs. normal cells]. No changes in intracellular synthesis of GnRH were noted [1,400 ± 371 and 1,395 ± 384 pg GnRH/4 × 106 cells for antisense and sense oligo(dN), respectively]. Antisense oligo(dN), but not sense or missense oligo(dN), inhibited cAMP-dependent36Cl efflux. The expression of CFTR protein, detected by Western blotting, was also inhibited 68% by preincubation of cells with antisense oligo(dN). GT1–7 hypothalamic neurons express the CFTR gene, mRNA, and protein, which modulate neurosecretion. Abnormal neuropeptide vesicle trafficking by mutant CFTR may help to explain some of the diverse manifestations of cystic fibrosis.

scholarly journals Unraveling the Mechanism of a LOV Domain Optogenetic Sensor: A Glutamine Lever Induces Unfolding of the Jα Helix

2020 ◽  
Author(s):  
James N. Iuliano ◽  
Jinnette Tolentino Collado ◽  
Agnieszka A. Gil ◽  
Pavithran T. Ravindran ◽  
Andras Lukacs ◽  
...  
Keyword(s):  
Structural Dynamics ◽  
Conformational Changes ◽  
Interdisciplinary Approach ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Lov Domain ◽  
Time Resolved ◽  
Protein Structural Dynamics ◽  
Dynamics Simulations ◽  
Time Resolved Infrared Spectroscopy

AbstractLight-activated protein domains provide a convenient, modular, and genetically encodable sensor for optogenetics and optobiology. Although these domains have now been deployed in numerous systems, the precise mechanism of photoactivation and the accompanying structural dynamics that modulate output domain activity remain to be fully elucidated. In the C-terminal light, oxygen, voltage (LOV) domain of plant phototropins (LOV2), blue light activation leads to formation of an adduct between a conserved Cys residue and the embedded FMN chromophore, rotation of a conserved Gln (Q513), and unfolding of a helix (Jα-helix) which is coupled to the output partner. In the present work, we focus on the allosteric pathways leading to Jα helix unfolding in Avena sativa LOV2 (AsLOV2) using an interdisciplinary approach involving molecular dynamics simulations extending to 7 μs, time-resolved infrared spectroscopy, solution NMR spectroscopy, and in-cell optogenetic experiments. In the dark state, the side chain of N414 is hydrogen bonded to the backbone N-H of Q513. The simulations predict a lever-like motion of Q513 after Cys adduct formation resulting in loss of the interaction between the side chain of N414 and the backbone C=O of Q513, and formation of a transient hydrogen bond between the Q513 and N414 side chains. The central role of N414 in signal transduction was evaluated by site-directed mutagenesis supporting a direct link between Jα helix unfolding dynamics and the cellular function of the Zdk2-AsLOV2 optogenetic construct. Through this multifaceted approach, we show that Q513 and N414 are critical mediators of protein structural dynamics, linking the ultrafast (sub-ps) excitation of the FMN chromophore to the microsecond conformational changes that result in photoreceptor activation and biological function.

scholarly journals Full Rescue of F508del-CFTR Processing and Function by CFTR Modulators can be Achieved by Removal of Two Unique Regulatory Regions

2018 ◽  
Author(s):  
Inna Uliyakina ◽  
Ana C Da Paula ◽  
Sara Afonso ◽  
Miguel J Lobo ◽  
Verónica Felício ◽  
...  
Keyword(s):  
Negative Impact ◽  
Site Directed Mutagenesis ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Channel Opening ◽  
Cell Surface Biotinylation ◽  
Cf Transmembrane Conductance Regulator ◽  
And Function ◽  
The Impact ◽  
Cftr Modulators

AbstractBackground and Purpose: Cystic Fibrosis (CF) is caused by mutations in the CF Transmembrane conductance Regulator (CFTR), the only ABC transporter functioning as a channel. Unique to CFTR are two highly conformationally dynamic regions: the regulatory extension (RE) and regulatory insertion (RI). Removal of the latter rescues the trafficking defect of CFTR with F508del, the most common CF-causing mutation.We aimed here to assess the impact of RE removal (alone or with RI or genetic revertants) on F508del-CFTR traffic and how CFTR modulator drugs corrector VX-809/lumacaftor and potentiator VX-770/ivacaftor rescue these combined variants so as to gain insight into the mechanism of action (MoA) of these drugs.Experimental Approach. We generated ∆RE and ∆RI CFTR variants (with and without genetic revertants) by site-directed mutagenesis and used them to stably transfect BHK cell lines. We studied CFTR expression and stability by Western blotting and pulse-chase respectively, plasma membrane levels by cell surface biotinylation and channel activity by the iodide efflux technique.Key Results. Our data demonstrate that ∆RI significantly enhanced rescue of F508del-CFTR by VX-809. Thus, while the presence of the regulatory insertion seems to be precluding full rescue of F508del-CFTR processing by VX-809, this region appears essential to rescue its function by VX-770, thus suggesting some contradictory role in rescue of F508del-CFTR by these two modulators. Nevertheless, this negative impact of RI removal on VX-770-stimulated currents on F508del-CFTR can be compensated by deletion of the regulatory extension which also leads to the stabilization of this mutant. We thus propose that, despite both these regions being conformationally active, RI precludes F508del-CFTR processing while RE affects mostly its stability and channel opening.Supporting Information: Additional figures with supplementary data

scholarly journals Real Life Evaluation of the Multi-organ Effects of Lumacaftor/Ivacaftor on F508del Homozygous Cystic Fibrosis Patients

2021 ◽  
Author(s):  
Karin Yaacoby-Bianu ◽  
Zeev Schnapp ◽  
Ilana Koren ◽  
Anat Ilivitzki ◽  
Mohamed Khatib ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Mineral Metabolism ◽  
Real Life ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Sweat Chloride ◽  
Registration Number ◽  
Cftr Protein ◽  
One Year ◽  
And Function

Abstract Background: Lumacaftor/Ivacaftor (LUM-IVA), a cystic fibrosis transmembrane conductance regulator (CFTR) protein corrector-potentiator combination, improves lung function and reduces pulmonary exacerbations (PEx) in F508del homozygous CF patients. However, the systemic effects of LUM-IVA outside the respiratory and nutritional domains have not yet been thoroughly investigated.Methods: A prospective, real-world, one-year study was performed on F508del homozygous adult CF patients who commenced treatment with LUM-IVA. Pancreatic function, bone metabolism, fertility status, nutritional and pulmonary factors were evaluated. Results: 12 patients with a mean age of 28.3 years (18.6-43.9) were recruited. Following 12 months of treatment, no changes were detected in glucose, insulin, c-peptide or BMI values. A significant relative decrease in mean alkaline-phosphatase levels (122.8 U/L vs 108, p=0.008) and a trend toward an increase in calcium levels (9.5 vs 9.8 mg/dL, p=0.0681) were observed. A non-significant improvement in mean DEXA spine t-score after a year of treatment (-2.1 vs -1.6, n=4, p=0.12) was detected. Sweat chloride concentrations decreased significantly after treatment (-21.4 mEq/L; p=0.003). Pulmonary outcome evaluations revealed improvement in spirometry values during the first three months (FEV1 by 5.7% p=0.019, FEF25-75 by 4.3% p=0.035) with no change in chest CT Bhalla score and CFQR after one year. There was also a shift from IV to oral antibiotics for PEx treatment.Conclusions: After a year of treatment, a stabilization was observed in the pancreatic indices, nutritional status, structure and function of the lungs, with a beneficial effect on bone mineral metabolism and CFTR function. Additional studies should investigate the effect of CFTR modulators on extra-pulmonary manifestations.The ClinicalTrials.gov registration number of the study is NCT04623879, registered on 10/11/2020, “retrospectively registered”.

Influence of Flexible Side-Chains on the Breathing Phase Transition of Pillared Layer MOFs: A Force Field Investigation

2020 ◽  
Author(s):  
Julian Keupp ◽  
Johannes P. Dürholt ◽  
Rochus Schmid
Keyword(s):  
First Principles ◽  
Good Accuracy ◽  
Field Investigation ◽  
Square Lattice ◽  
Side Chain ◽  
Second Step ◽  
Side Chains ◽  
Dynamics Simulations ◽  
Complex Phenomena ◽  
Closed Pore

The prototypical pillared layer MOFs, formed by a square lattice of paddle-<br>wheel units and connected by dinitrogen pillars, can undergo a breathing phase<br>transition by a “wine-rack” type motion of the square lattice. We studied this not<br>yet fully understood behavior using an accurate first principles parameterized force<br>field (MOF-FF) for larger nanocrystallites on the example of Zn 2 (bdc) 2 (dabco) [bdc:<br>benzenedicarboxylate, dabco: (1,4-diazabicyclo[2.2.2]octane)] and found clear indi-<br>cations for an interface between a closed and an open pore phase traveling through<br>the system during the phase transformation [Adv. Theory Simul. 2019, 2, 11]. In<br>conventional simulations in small supercells this mechanism is prevented by periodic<br>boundary conditions (PBC), enforcing a synchronous transformation of the entire<br>crystal. Here, we extend this investigation to pillared layer MOFs with flexible<br>side-chains, attached to the linker. Such functionalized (fu-)MOFs are experimen-<br>tally known to have different properties with the side-chains acting as fixed guest<br>molecules. First, in order to extend the parameterization for such flexible groups,<br>1a new parametrization strategy for MOF-FF had to be developed, using a multi-<br>structure force based fit method. The resulting parametrization for a library of<br>fu-MOFs is then validated with respect to a set of reference systems and shows very<br>good accuracy. In the second step, a series of fu-MOFs with increasing side-chain<br>length is studied with respect to the influence of the side-chains on the breathing<br>behavior. For small supercells in PBC a systematic trend of the closed pore volume<br>with the chain length is observed. However, for a nanocrystallite model a distinct<br>interface between a closed and an open pore phase is visible only for the short chain<br>length, whereas for longer chains the interface broadens and a nearly concerted trans-<br>formation is observed. Only by molecular dynamics simulations using accurate force<br>fields such complex phenomena can be studied on a molecular level.

Modeling the Effects of O-sulfonation on the CID of Serine

2020 ◽  
Author(s):  
Kenneth Lucas ◽  
George Barnes
Keyword(s):  
Sulfo Group ◽  
High Energy ◽  
Empirical Method ◽  
Side Chain ◽  
Energy Structure ◽  
Direct Dynamics ◽  
Theoretical Mass ◽  
Intermolecular Proton Transfer ◽  
Dynamics Simulations ◽  
Semi Empirical

We present the results of direct dynamics simulations and DFT calculations aimed at elucidating the effect of \textit{O}-sulfonation on the collision induced dissociation for serine. Towards this end, direct dynamics simulations of both serine and sulfoserine were performed at multiple collision energies and theoretical mass spectra obtained. Comparisons to experimental results are favorable for both systems. Peaks related to the sulfo group are identified and the reaction dynamics explored. In particular, three significant peaks (m\z 106, 88, and 81) seen in the theoretical mass spectrum directly related to the sulfo group are analyzed as well as major peaks shared by both systems. Our analysis shows that the m\z 106 peaks result from intramolecular rearrangements, intermolecular proton transfer among complexes composed of initial fragmentation products, and at high energy side-chain fragmentation. The \mz 88 peak was found to contain multiple constitutional isomers, including a previously unconsidered, low energy structure. It was also seen that the RM1 semi empirical method was not able to obtain all of the major peaks seen in experiment for sulfoserine. In contrast, PM6 did obtain all major experimental peaks.

Molecular Basis of Glucose Transport Mechanism in Plants

2019 ◽  
Author(s):  
Balaji Selvam ◽  
Ya-Chi Yu ◽  
Liqing Chen ◽  
Diwakar Shukla
Keyword(s):  
Molecular Dynamics ◽  
Free Energy ◽  
Molecular Dynamics Simulations ◽  
Conformational Changes ◽  
Transport Mechanism ◽  
Conformational Dynamics ◽  
Site Directed Mutagenesis ◽  
Free Energy Barrier ◽  
Transport Cycle ◽  
Dynamics Simulations

<p>The SWEET family belongs to a class of transporters in plants that undergoes large conformational changes to facilitate transport of sugar molecules across the cell membrane. However, the structures of their functionally relevant conformational states in the transport cycle have not been reported. In this study, we have characterized the conformational dynamics and complete transport cycle of glucose in OsSWEET2b transporter using extensive molecular dynamics simulations. Using Markov state models, we estimated the free energy barrier associated with different states as well as 1 for the glucose the transport mechanism. SWEETs undergoes structural transition to outward-facing (OF), Occluded (OC) and inward-facing (IF) and strongly support alternate access transport mechanism. The glucose diffuses freely from outside to inside the cell without causing major conformational changes which means that the conformations of glucose unbound and bound snapshots are exactly same for OF, OC and IF states. We identified a network of hydrophobic core residues at the center of the transporter that restricts the glucose entry to the cytoplasmic side and act as an intracellular hydrophobic gate. The mechanistic predictions from molecular dynamics simulations are validated using site-directed mutagenesis experiments. Our simulation also revealed hourglass like intermediate states making the pore radius narrower at the center. This work provides new fundamental insights into how substrate-transporter interactions actively change the free energy landscape of the transport cycle to facilitate enhanced transport activity.</p>

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