Gergely Lukacs - Profile on Academia.edu (original) (raw)

Papers by Gergely Lukacs

Additional file 1: of A Christianson syndrome-linked deletion mutation (∆287ES288) in SLC9A6 disrupts recycling endosomal function and elicits neurodegeneration and cell death

Supplementary data. (PDF 915 kb)

Structure of PINK1 reveals autophosphorylation dimer and provides insights into binding to the TOM complex

SummaryMutations in PINK1 causes autosomal-recessive Parkinson’s disease. Mitochondrial damage re... more SummaryMutations in PINK1 causes autosomal-recessive Parkinson’s disease. Mitochondrial damage results in PINK1 import arrest on the Translocase of the Outer Mitochondrial Membrane (TOM) complex, resulting in the activation of its ubiquitin kinase activity by autophosphorylation and initiation of Parkin-dependent mitochondrial clearance. Herein we report crystal structures of the entire cytosolic domain of insect PINK1. Our structures reveal a dimeric autophosphorylation complex targeting phosphorylation at the invariant Ser205 (human Ser228). The dimer interface requires insert 2, which is unique to PINK1. The structures also reveal how an N-terminal helix binds to the C-terminal extension and provide insights into stabilization of PINK1 on the core TOM complex.

Responsiveness of mutants of NHE1 isoform of Na+/H+ antiport to osmotic stress

American Journal of Physiology-Cell Physiology, 1995

Hypertonic activation of NHE1, the ubiquitous Na+/H+ exchanger, plays a central role in cell volu... more Hypertonic activation of NHE1, the ubiquitous Na+/H+ exchanger, plays a central role in cell volume regulation, yet little is known about the underlying mechanism. We probed the osmotic responsiveness of full-length and truncated constructs of NHE1 transfected into cells lacking endogenous antiport activity. The hypertonic stimulation of NHE1 was preserved after heterologous transfection of the full-length NHE1 or of constructs truncated at positions 698 or 703. In contrast, mutants truncated at position 635 (delta 635) failed to respond to osmotic challenge. Transfectants (delta 635) behaved as if constitutively activated, having a permanently elevated cytosolic pH (pHi) under isotonic, unstimulated conditions. The delta 635 mutant displayed H+ binding with high affinity and low cooperativity. Constructs delta 582 or delta 566 had a reduced H+ sensitivity and were therefore inactive at resting pHi. Such cells were unresponsive to osmotic stress near physiological pHi but could be a...

Proton conductance of the plasma membrane: properties, regulation, and functional role

American Journal of Physiology-Cell Physiology, 1993

H+ conductive pathways have been detected in the plasma membranes of a variety of cell types. The... more H+ conductive pathways have been detected in the plasma membranes of a variety of cell types. The large exquisitely H(+)-selective permeability of the conductive pathway can support sizable net H+ fluxes. Although subtle differences exist among tissues and species, certain common features suggest that related transport systems are involved in all cases. The H+ conductance is gated by depolarizing voltages and is promoted by intracellular acidification. Conversely, extracellular acidification inhibits the conductance. These features facilitate net H+ efflux, while precluding potentially deleterious H+ uptake. In some cell types, activation of the conductance is additionally controlled by physiological ligands and by second messengers. The conductance most likely functions in the regulation of intracellular pH, contributing to the extrusion of H+ during repetitive depolarization of the plasma membrane, as occurs in neurons and muscle cells. This pathway may be particularly relevant in...

PREFERENTIAL SUMOYLATION OF Delta F508 CFTR AND NBD1 LEADS TO PROTEIN DEGRADATION

HSP27 SELECTIVELY TARGETS Delta F508 CFTR NBD1 FOR DEGRADATION VIA THE SUMO PATHWAY

Journal of Cell Biology, May 21, 2001

Impaired biosynthetic processing of the cys- tic fibrosis (CF) transmembrane conductance regulato... more Impaired biosynthetic processing of the cys- tic fibrosis (CF) transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel, constitutes the most common cause of CF. Recently, we have identified a distinct category of mutation, caused by premature stop codons and frameshift mutations, which manifests in diminished expression of COOH-terminally truncated CFTR at the cell surface. Although the biosynthetic processing and plasma membrane targeting of truncated CFTRs are preserved, the turnover of the complex-glycosylated mutant is sixfold faster than its wild-type (wt) counterpart. Destabilization of the truncated CFTR coincides with its enhanced susceptibility to proteasome-dependent degradation from post-Golgi compartments globally, and the plasma membrane specifically, determined by pulse-chase analysis in conjunction with cell surface biotinylation. Proteolytic cleavage of the full-length complex-glycosylated wt and degradation intermediates derived from both T70 and wt CFTR requires endolysosomal proteases. The enhanced protease sensitivity in vitro and the decreased thermostability of the complex-glycosylated T70 CFTR in vivo suggest that structural destabilization may account for the increased proteasome susceptibility and the short residence time at the cell surface. These in turn are responsible, at least in part, for the phenotypic manifestation of CF. We propose that the proteasome-ubiquitin pathway may be involved in the peripheral quality control of other, partially unfolded membrane proteins as well.

Reviews of Physiology Biochemistry and Pharmacology, 2016

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that conducts chlori... more Cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that conducts chloride and bicarbonate ions across epithelial cell membranes. Mutations in the CFTR gene diminish the ion channel function and lead to impaired epithelial fluid transport in multiple organs such as the lung and the pancreas resulting in cystic fibrosis. Heterozygous carriers of CFTR mutations do not develop cystic fibrosis but exhibit increased risk for pancreatitis and associated pancreatic damage characterized by elevated mucus levels, fibrosis and cyst formation. Importantly, recent studies demonstrated that pancreatitis causing insults, such as alcohol, smoking or bile acids strongly inhibit CFTR function. Furthermore, human studies showed reduced levels of CFTR expression and function in all forms of pancreatitis. These findings indicate that impairment of CFTR is critical in the development of pancreatitis; therefore, correcting CFTR function could be the first specific therapy in pancreatitis. In this review, we summarize recent advances in the field and discuss new possibilities for the treatment of pancreatitis.

Nature Structural & Molecular Biology, Jul 19, 2018

In the version of this article initially published, RING2 in the schematic to the left in Fig. wa... more In the version of this article initially published, RING2 in the schematic to the left in Fig. was mislabeled as RING0. The error has been corrected in the HTML and PDF versions of the article.

Humana Press eBooks, 2011

The wild-type CFTR channel undergoes constitutive internalization and recycling at the plasma mem... more The wild-type CFTR channel undergoes constitutive internalization and recycling at the plasma membrane. This process is initiated by the recognition of the Tyr-and di-Leu-based endocytic motifs of CFTR by the AP-2 adaptor complex, leading to the formation of clathrin-coated vesicles and the channel delivery to sorting/recycling endosomes. Accumulating evidence suggests that conformationally defective mutant CFTRs (e.g. rescued ΔF508 and glycosylation-deficient channel) are unstable at the plasma membrane and undergo augmented ubiquitination in post-Golgi compartments. Ubiquitination conceivably accounts for the metabolic instability at cell surface by provoking accelerated internalization, as well as rerouting the channel from recycling towards lysosomal degradation. We developed an in vivo fluorescence ratio imaging assay (FRIA) that in concert with genetic manipulation can be utilized to establish the post-endocytic fate and sorting determinants of mutant CFTRs.

American Journal of Physiology-lung Cellular and Molecular Physiology, Apr 1, 2018

The EGF receptor (EGFR)/a disintegrin and metalloproteinase 17 (ADAM17) signaling pathway mediate... more The EGF receptor (EGFR)/a disintegrin and metalloproteinase 17 (ADAM17) signaling pathway mediates the shedding of growth factors and secretion of cytokines and is involved in chronic inflammation and tissue remodeling. Since these are hallmarks of cystic fibrosis (CF) lung disease, we hypothesized that CF transmembrane conductance regulator (CFTR) deficiency enhances EGFR/ADAM17 activity in human bronchial epithelial cells. In CF bronchial epithelial CFBE41o Ϫ cells lacking functional CFTR (iCFTR Ϫ ) cultured at air-liquid interface (ALI) we found enhanced ADAM17-mediated shedding of the EGFR ligand amphiregulin (AREG) compared with genetically identical cells with induced CFTR expression (iCFTR ϩ ). Expression of the inactive G551D-CFTR did not have this effect, suggesting that active CFTR reduces EGFR/ADAM17 activity. This was confirmed in CF compared with normal differentiated primary human bronchial epithelial cells (HBEC-ALI). ADAM17-mediated AREG shedding was tightly regulated by the EGFR/MAPK pathway. Compared with iCFTR ϩ cells, iCFTR Ϫ cells displayed enhanced apical presentation and phosphorylation of EGFR, in accordance with enhanced EGFR/ ADAM17 activity in CFTR-deficient cells. The nonpermeant natural antioxidant glutathione (GSH) strongly inhibited AREG release in iCFTR and in primary HBEC-ALI, suggesting that ADAM17 activity is directly controlled by extracellular redox potentials in differentiated airway epithelium. Furthermore, the fluorescent redox probe glutaredoxin 1-redox-sensitive green fluorescent protein-glycosylphosphatidylinositol (Grx1-roGFP-GPI) indicated more oxidized conditions in the extracellular space of iCFTR Ϫ cells, consistent with the role of CFTR in GSH transport. Our data suggest that in CFTR-deficient airway epithelial cells a more oxidized state of the extracellular membrane, likely caused by defective GSH secretion, leads to enhanced activity of the EGFR/ADAM17 signaling axis.

The Journal of Physiology, Oct 9, 2017

Characterisation of most mutations found in CLCN2 in patients with CC2L leukodystrophy show that ... more Characterisation of most mutations found in CLCN2 in patients with CC2L leukodystrophy show that they cause a reduction in function of the chloride channel ClC-2. r GlialCAM, a regulatory subunit of ClC-2 in glial cells and involved in the leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts (MLC), increases the activity of a ClC-2 mutant by affecting ClC-2 gating and by stabilising the mutant at the plasma membrane. r The stabilisation of ClC-2 at the plasma membrane by GlialCAM depends on its localisation at cell-cell junctions. r The membrane protein MLC1, which is defective in MLC, also contributes to the stabilisation of ClC-2 at the plasma membrane, providing further support for the view that GlialCAM, MLC1 and ClC-2 form a protein complex in glial cells.

Journal of Biological Chemistry, Jul 1, 2017

Edited by Norma Allewell The enzyme UDP-glucose:glycoprotein glucosyltransferase (UGGT) mediates ... more Edited by Norma Allewell The enzyme UDP-glucose:glycoprotein glucosyltransferase (UGGT) mediates quality control of glycoproteins in the endoplasmic reticulum by attaching glucose to N-linked glycan of misfolded proteins. As a sensor, UGGT ensures that misfolded proteins are recognized by the lectin chaperones and do not leave the secretory pathway. The structure of UGGT and the mechanism of its selectivity for misfolded proteins have been unknown for 25 years. Here, we used negative-stain electron microscopy and small-angle X-ray scattering to determine the structure of UGGT from Drosophila melanogaster at 18-Å resolution. Three-dimensional reconstructions revealed a cagelike structure with a large central cavity. Particle classification revealed flexibility that precluded determination of a highresolution structure. Introduction of biotinylation sites into a fungal UGGT expressed in Escherichia coli allowed identification of the catalytic and first thioredoxin-like domains. We also used hydrogen-deuterium exchange mass spectrometry to map the binding site of an accessory protein, Sep15, to the first thioredoxin-like domain. The UGGT structural features identified suggest that the central cavity contains the catalytic site and is lined with hydrophobic surfaces. This enhances the binding of misfolded substrates with exposed hydrophobic residues and excludes folded proteins with hydrophilic surfaces. In conclusion, we have determined the UGGT structure, which enabled us to develop a plausible functional model of the mechanism for UGGT's selectivity for misfolded glycoproteins.

Molecular Pharmacology, Apr 15, 2014

The most prevalent cystic fibrosis transmembrane conductance regulator (CFTR) mutation causing cy... more The most prevalent cystic fibrosis transmembrane conductance regulator (CFTR) mutation causing cystic fibrosis, DF508, impairs folding of nucleotide binding domain (NBD) 1 and stability of the interface between NBD1 and the membranespanning domains. The interfacial stability defect can be partially corrected by the investigational drug )cyclopropyl]carbonyl]amino]-3methyl-2-pyridinyl]-benzoic acid) or the R1070W mutation. Second-generation DF508-CFTR correctors are needed to improve on the modest efficacy of existing cystic fibrosis correctors. We postulated that a second corrector targeting a distinct folding/interfacial defect might act in synergy with VX-809 or the R1070W suppressor mutation. A biochemical screen for DF508-CFTR cell surface expression was developed in a human lung epithelium-derived cell line (CFBE41o 2 ) by expressing chimeric CFTRs with a horseradish peroxidase (HRP) in the fourth exofacial loop in either the presence or absence of R1070W. Using a luminescence readout of HRP activity, screening of approximately 110,000 small molecules produced nine novel corrector scaffolds that increased cell surface ΔF508-CFTR expression by up to 200% in the presence versus absence of maximal VX-809. Further screening of 1006 analogs of compounds identified from the primary screen produced 15 correctors with an EC 50 , 5 mM. Eight chemical scaffolds showed synergy with VX-809 in restoring chloride permeability in ΔF508-expressing A549 cells. An aminothiazole increased chloride conductance in human bronchial epithelial cells from a DF508 homozygous subject beyond that of maximal VX-809. Mechanistic studies suggested that NBD2 is required for the aminothiazole rescue. Our results provide proof of concept for synergy screening to identify second-generation correctors, which, when used in combination, may overcome the "therapeutic ceiling" of first-generation correctors.

Journal of Biological Chemistry, 2016

Cellular and Molecular Life Sciences, 2022

Transmembrane (TM) proteins are major drug targets, but their structure determination, a prerequi... more Transmembrane (TM) proteins are major drug targets, but their structure determination, a prerequisite for rational drug design, remains challenging. Recently, the DeepMind's AlphaFold2 machine learning method greatly expanded the structural coverage of sequences with high accuracy. Since the employed algorithm did not take specific properties of TM proteins into account, the reliability of the generated TM structures should be assessed. Therefore, we quantitatively investigated the quality of structures at genome scales, at the level of ABC protein superfamily folds and for specific membrane proteins (e.g. dimer modeling and stability in molecular dynamics simulations). We tested template-free structure prediction with a challenging TM CASP14 target and several TM protein structures published after AlphaFold2 training. Our results suggest that AlphaFold2 performs well in the case of TM proteins and its neural network is not overfitted. We conclude that cautious applications of AlphaFold2 structural models will advance TM protein-associated studies at an unexpected level.

$Research paper thumbnail of Binding of inositol phosphates and induction of Ca2+ release from pituitary microsomal fractions$

Biochemical Journal, Jun 1, 1987

Bovine anterior-pituitary microsomal fractions exhibit high-affinity, saturable and reversible bi... more Bovine anterior-pituitary microsomal fractions exhibit high-affinity, saturable and reversible binding of inositol 1,4,5-[32P]trisphosphate; 50%O of the labelled ligand is displaced by 3.5 nM-inositol 1,4,5- trisphosphate, 0.5/tM-inositol 1,4-bisphosphate and 10 gM-ATP. Inositol 1,4,5-trisphosphate induces the release of Ca2+ from the microsomal vesicles (half-maximal effect at 290 nM), and its action is potentiated by inositol tetrakisphosphate (half-maximal effect at 4,UM).

Molecular Biology of the Cell, Apr 1, 2009

The cystic fibrosis transmembrane conductance regulator (CFTR) architecture consists of two membr... more The cystic fibrosis transmembrane conductance regulator (CFTR) architecture consists of two membrane spanning domains (MSD1 and -2), two nucleotide binding domains (NBD1 and -2), and a regulatory (R) domain. Several point mutations lead to the channel misprocessing, with limited structural perturbation of the mutant domain. To gain more insight into the basis of CFTR folding defect, the contribution of domain-wise and cooperative domain folding was assessed by determining 1) the minimal domain combination that is recognized as native and can efficiently escape the endoplasmic reticulum (ER) retention and 2) the impact of mutation on the conformational coupling among domains. One-, two-, three-, and most of the four-domain assemblies were retained at the ER. Solubilization mutations, however, rescued the NBD1 processing defect conceivably by thermodynamic stabilization. The smallest folding unit that traversed the secretory pathway was composed of MSD1-NBD1-R-MSD2 as a linear or split polypeptide. Cystic fibrosis-causing missense mutations in the MSD1, NBD1, MSD2, and NBD2 caused conformational defect in multiple domains. We propose that cooperative posttranslational folding is required for domain stabilization and provides a plausible explanation for the global misfolding caused by point mutations dispersed along the full-length CFTR.

Scientific Reports, Sep 3, 2019

Genetic and acquired loss-of-function defect of the cystic fibrosis transmembrane conductance reg... more 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 cAMPdependent 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. The adverse long-term effects of inhaled, combusted tobacco or cigarette smoke (CS) on the lung's cellular and molecular physiology have been established and include DNA damage, goblet cell metaplasia, increased inflammation, autophagy and proteolysis 1 . These processes account for higher incidence of chronic obstructive pulmonary disease (COPD), the third leading cause of death in the US 2 , which is compounded by significant extrapulmonary pathologies 1,3 and lung cancer. The acquired loss of the cystic fibrosis transmembrane conductance regulator (CFTR) function, a chloride and bicarbonate-selective, 3′,5′-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) regulated anion channel 4 , is invoked in the pathogenesis of COPD partly as a consequence of CS exposure . COPD exhibits hallmarks of chronic bronchitis and emphysema, with overlapping clinical manifestation and molecular pathology to that of cystic fibrosis (CF) . Both acquired and inherited expression defects of CFTR result in the periciliary liquid layer depletion, acidification, mucus dehydration, increased bacterial adhesion, and decreased mucociliary clearance (MCC), causing recurrent infections and sustained inflammation with progressive deterioration of the lung tissue 6,9-12 . Compelling evidence indicates that subacute (2-24 h) and chronic (>24 h) cigarette smoke (CS) exposure compromises CFTR activity at the protein and mRNA level in a variety of cell types, including human nasal and bronchial epithelia . In contrast, the acute effect of CS on CFTR is incompletely understood. CFTR, a member of the adenosine triphosphate (ATP)-binding cassette transporter (ABC) superfamily, is predominantly localised at the apical plasma membrane (PM) of secretory and resorptive epithelia in various organs,