Keratin 8 is a scaffolding and regulatory protein of ERAD complexes.

Early recognition and enhanced degradation of misfolded proteins by the endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD) cause defective protein secretion and membrane targeting, as exemplified for Z-alpha-1-antitrypsin (Z-A1AT), responsible for alpha-1-antitrypsin deficiency (A1ATD) and F508del-CFTR (cystic fibrosis transmembrane conductance regulator) responsible for cystic fibrosis (CF). Prompted by our previous observation that decreasing Keratin 8 (K8) expression increased trafficking of F508del-CFTR to the plasma membrane, we investigated whether K8 impacts trafficking of soluble misfolded Z-A1AT protein. The subsequent goal of this study was to elucidate the mechanism underlying the K8-dependent regulation of protein trafficking, focusing on the ERAD pathway. The results show that diminishing K8 concentration in HeLa cells enhances secretion of both Z-A1AT and wild-type (WT) A1AT with a 13-fold and fourfold increase, respectively. K8 down-regulation triggers ER failure and cellular apoptosis when ER stress is jointly elicited by conditional expression of the µs heavy chains, as previously shown for Hrd1 knock-out. Simultaneous K8 silencing and Hrd1 knock-out did not show any synergistic effect, consistent with K8 acting in the Hrd1-governed ERAD step. Fractionation and co-immunoprecipitation experiments reveal that K8 is recruited to ERAD complexes containing Derlin2, Sel1 and Hrd1 proteins upon expression of Z/WT-A1AT and F508del-CFTR. Treatment of the cells with c407, a small molecule inhibiting K8 interaction, decreases K8 and Derlin2 recruitment to high-order ERAD complexes. This was associated with increased Z-A1AT secretion in both HeLa and Z-homozygous A1ATD patients' respiratory cells. Overall, we provide evidence that K8 acts as an ERAD modulator. It may play a scaffolding protein role for early-stage ERAD complexes, regulating Hrd1-governed retrotranslocation initiation/ubiquitination processes. Targeting K8-containing ERAD complexes is an attractive strategy for the pharmacotherapy of A1ATD.

Structural Dynamics of the Vimentin Coiled-coil Contact Regions Involved in Filament Assembly as Revealed by Hydrogen-Deuterium Exchange.

Intermediate filaments (IF) are major constituents of the cytoskeleton of metazoan cells. They are not only responsible for the mechanical properties but also for various physiological activities in different cells and tissues. The building blocks of IFs are extended coiled-coil-forming proteins exhibiting a characteristic central α-helical domain ("rod"). The fundamental principles of the filament assembly mechanism and the network formation have been widely elucidated for the cytoplasmic IF protein vimentin. Also, a comprehensive structural model for the tetrameric complex of vimentin has been obtained by X-ray crystallography in combination with various biochemical and biophysical techniques. To extend these static data and to investigate the dynamic properties of the full-length proteins in solution during the various assembly steps, we analyzed the patterns of hydrogen-deuterium exchange in vimentin and in four variants carrying point mutations in the IF consensus motifs present at either end of the α-helical rod that cause an assembly arrest at the unit-length filament (ULF) stage. The results yielded unique insights into the structural properties of subdomains within the full-length vimentin, in particular in regions of contact in α-helical and linker segments that stabilize different oligomeric forms such as tetramers, ULFs, and mature filaments. Moreover, hydrogen-deuterium exchange analysis of the point-mutated variants directly demonstrated the active role of the IF consensus motifs in the oligomerization mechanism of tetramers during ULF formation. Ultimately, using molecular dynamics simulation procedures, we provide a structural model for the subdomain-mediated tetramer/tetramer interaction via "cross-coiling" as the first step of the assembly process.

Analysis of distinct molecular assembly complexes of keratin K8 and K18 by hydrogen-deuterium exchange.

Keratins are intermediate filament (IF) proteins that form complex filament systems in epithelial cells, thus serving as scaffolding elements and mechanical stress absorbers. The building blocks of keratin IFs are parallel coiled-coil dimers of two distinct sequence-related proteins distinguished as type I and type II keratins. To gain more insight into their structural dynamics, we resorted to hydrogen-deuterium exchange mass spectrometry of keratins K8 and K18, which are characteristic for simple epithelial cells. Using this powerful technique not employed with IFs before, we mapped patterns of protected versus unprotected regions in keratin complexes at various assembly levels. In particular, we localized protein segments exhibiting different hydrogen exchange patterns in tetramers versus filaments. We observed a general pattern of precisely positioned regions of stability intertwining with flexible regions, mostly represented by the non-α-helical segments. Notably, some regions within the coiled-coil domains are significantly more dynamic than others, while the IF-consensus motifs at the end domains of the central α-helical "rod" segment, which mediate the "head-to-tail" dimer-dimer interaction in the filament elongation process, become distinctly more protected upon formation of filaments. Moreover, to gain more insight into the dynamics of the individual keratins, we investigated the properties of homomeric preparations of K8 and K18. The physiological importance of keratins without a partner is encountered in both pathological and experimental situations when one of the two species is present in robust excess or completely absent, such as in gene-targeted mice.

Readthrough-induced misincorporated amino acid ratios guide mutant-specific therapeutic approaches for two CFTR nonsense mutations.

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 understanding of molecular defects underlying various CFTR nonsense mutations and provide a foundation to refine mutation-dependent therapeutic strategies for various CF-causing nonsense mutations.

Folding correctors can restore CFTR posttranslational folding landscape by allosteric domain-domain coupling.

The folding/misfolding and pharmacological rescue of multidomain ATP-binding cassette (ABC) C-subfamily transporters, essential for organismal health, remain incompletely understood. The ABCC transporters core consists of two nucleotide binding domains (NBD1,2) and transmembrane domains (TMD1,2). Using molecular dynamic simulations, biochemical and hydrogen deuterium exchange approaches, we show that the mutational uncoupling or stabilization of NBD1-TMD1/2 interfaces can compromise or facilitate the CFTR(ABCC7)-, MRP1(ABCC1)-, and ABCC6-transporters posttranslational coupled domain-folding in the endoplasmic reticulum. Allosteric or orthosteric binding of VX-809 and/or VX-445 folding correctors to TMD1/2 can rescue kinetically trapped CFTR posttranslational folding intermediates of cystic fibrosis (CF) mutants of NBD1 or TMD1 by global rewiring inter-domain allosteric-networks. We propose that dynamic allosteric domain-domain communications not only regulate ABCC-transporters function but are indispensable to tune the folding landscape of their posttranslational intermediates. These allosteric networks can be compromised by CF-mutations, and reinstated by correctors, offering a framework for mechanistic understanding of ABCC-transporters (mis)folding.

Folding correctors can restore CFTR posttranslational folding landscape by allosteric domain-domain coupling.

The folding/misfolding and pharmacological rescue of multidomain ATP-binding cassette (ABC) C-subfamily transporters, essential for organismal health, remain incompletely understood. The ABCC transporters core consists of two nucleotide binding domains (NBD1,2) and transmembrane domains (TMD1,2). Using molecular dynamic simulations, biochemical and hydrogen deuterium exchange approaches, we show that the mutational uncoupling or stabilization of NBD1-TMD1/2 interfaces can compromise or facilitate the CFTR(ABCC7)-, MRP1(ABCC1)-, and ABCC6-transporters posttranslational coupled domain-folding in the endoplasmic reticulum. Allosteric or orthosteric binding of VX-809 and/or VX-445 folding correctors to TMD1/2 can rescue kinetically trapped CFTR post-translational folding intermediates of cystic fibrosis (CF) mutants of NBD1 or TMD1 by global rewiring inter-domain allosteric-networks. We propose that dynamic allosteric domain-domain communications not only regulate ABCC-transporters function but are indispensable to tune the folding landscape of their post-translational intermediates. These allosteric networks can be compromised by CF-mutations, and reinstated by correctors, offering a framework for mechanistic understanding of ABCC-transporters (mis)folding. One-Sentence Summary: Allosteric interdomain communication and its modulation are critical determinants of ABCC-transporters post-translational conformational biogenesis, misfolding, and pharmacological rescue.

A PI3Kγ mimetic peptide triggers CFTR gating, bronchodilation, and reduced inflammation in obstructive airway diseases.

Cyclic adenosine 3',5'-monophosphate (cAMP)-elevating agents, such as ß2-adrenergic receptor (ß2-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 ß2-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 asthma. In human bronchial epithelial cells, PI3Kγ MP induced unexpected cAMP and PKA elevations restricted to the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR), the ion channel controlling mucus hydration that is mutated in cystic fibrosis (CF). PI3Kγ MP promoted the phosphorylation of wild-type CFTR on serine-737, triggering channel gating, and rescued the function of F508del-CFTR, the most prevalent CF mutant, by enhancing the effects of existing CFTR modulators. These results unveil PI3Kγ as the regulator of a ß2-AR/cAMP microdomain central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways, suggesting the use of a PI3Kγ MP for compartment-restricted, therapeutic cAMP elevation in chronic obstructive respiratory diseases.

Mutation-specific dual potentiators maximize rescue of CFTR gating mutants.

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.

Phosphorylation-dependent modulation of CFTR macromolecular signalling complex activity by cigarette smoke condensate in airway epithelia.

Genetic and acquired loss-of-function defect of the cystic fibrosis transmembrane conductance regulator (CFTR) compromise airway surface liquid homeostasis and mucociliary clearance (MCC), culminating in recurrent lung inflammation/infection. While chronic cigarette smoke (CS), CS extract (CSE; water-soluble compounds) and CS condensate (CSC; particulate, organic fraction) exposure inhibit CFTR activity at transcriptional, biochemical, and functional levels, the acute impact of CSC remains incompletely understood. We report that CSC transiently activates CFTR chloride secretion in airway epithelia. The comparable CFTR phospho-occupancy after CSC- and forskolin-exposure, determined by affinity-enriched tandem mass spectrometry and pharmacology, suggest that localised cAMP-dependent protein kinase (PKA) stimulation by CSC causes the channel opening. Due to the inhibition of the MRP4/ABCC4, a cAMP-exporter confined to the CFTR macromolecular signalling-complex, PKA activation is accomplished by the subcompartmentalised elevation of cytosolic cAMP. In line, MRP4 inhibition results in CFTR activation and phospho-occupancy similar to that by forskolin. In contrast, acute CSC exposure reversibly inhibits the phosphorylated CFTR both in vivo and in phospholipid bilayers, without altering its cell surface density and phospho-occupancy. We propose that components of CSC elicit both a transient protective CFTR activation, as well as subsequent channel block in airway epithelia, contributing to the subacute MCC defect in acquired CF lung diseases.

New insights into interactions between the nucleotide-binding domain of CFTR and keratin 8.

The intermediate filament protein keratin 8 (K8) interacts with the nucleotide-binding domain 1 (NBD1) of the cystic fibrosis (CF) transmembrane regulator (CFTR) with phenylalanine 508 deletion (ΔF508), and this interaction hampers the biogenesis of functional ΔF508-CFTR and its insertion into the plasma membrane. Interruption of this interaction may constitute a new therapeutic target for CF patients bearing the ΔF508 mutation. Here, we aimed to determine the binding surface between these two proteins, to facilitate the design of the interaction inhibitors. To identify the NBD1 fragments perturbed by the ΔF508 mutation, we used hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) on recombinant wild-type (wt) NBD1 and ΔF508-NBD1 of CFTR. We then performed the same analysis in the presence of a peptide from the K8 head domain, and extended this investigation using bioinformatics procedures and surface plasmon resonance, which revealed regions affected by the peptide binding in both wt-NBD1 and ΔF508-NBD1. Finally, we performed HDX-MS analysis of the NBD1 molecules and full-length K8, revealing hydrogen-bonding network changes accompanying complex formation. In conclusion, we have localized a region in the head segment of K8 that participates in its binding to NBD1. Our data also confirm the stronger binding of K8 to ΔF508-NBD1, which is supported by an additional binding site located in the vicinity of the ΔF508 mutation in NBD1.

Rattlesnake Phospholipase A2 Increases CFTR-Chloride Channel Current and Corrects ∆F508CFTR Dysfunction: Impact in Cystic Fibrosis.

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 Xenopus laevis 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 ABCß 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.