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1.
Am J Physiol Lung Cell Mol Physiol ; 310(10): L993-L1002, 2016 05 15.
Article in English | MEDLINE | ID: mdl-26993521

ABSTRACT

MUC5B is a major polymeric mucin in the airway mucus gel and is an essential component of innate defense of the respiratory epithelium. Knowledge of the synthesis and intracellular processing of MUC5B is incomplete. We investigated the molecular details of MUC5B assembly in primary human bronchial epithelial cells (HBECs) grown at an air-liquid interface (ALI). Electrophoretic and centrifugal separations of intracellular forms of MUC5B probed with antibodies specific for non-O-glycosylated and O-glycosylated forms of the mucin identified three major intracellular populations of MUC5B (non-O-glycosylated monomer and dimer, and O-glycosylated polymers). Biophysical analysis of recombinant MUC5B COOH-terminus (CT5B; D4-B-C-CK) expressed in 293-EBNA cells showed that MUC5B dimerizes by disulfide linkage. Pulse-chase studies in the HBEC ALI cultures showed that non-O-glycosylated MUC5B was synthesized within 20 min of metabolic labeling and O-glycosylated, polymeric mucin within 2 h. Radiolabeled O-glycosylated mucin polymers were secreted within 2 h and the majority were released by 48 h. These data indicate that MUC5B follows a similar assembly to the related glycoprotein, von Willebrand factor (vWF); however, unlike vWF the MUC5B polypeptide shows no evidence of major proteolytic processing of D-domains during the production of the mature secreted polymeric mucin in normal and cystic fibrosis (CF) primary bronchial epithelial cells. In contrast, MUC5B D-domains were modified by neutrophil elastase, a protease commonly found in CF sputum, demonstrating that proteolytic degradation of MUC5B is an extracellular event in CF sputum. These results define the pathway for synthesis of MUC5B in primary human goblet cells.


Subject(s)
Mucin-5B/biosynthesis , Amino Acid Sequence , Cells, Cultured , Cystic Fibrosis/metabolism , Epithelial Cells/metabolism , Glycosylation , Humans , Leukocyte Elastase/chemistry , Mucin-5B/chemistry , Mucin-5B/genetics , Protein Processing, Post-Translational , Proteolysis
2.
J Neurochem ; 116(3): 342-9, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21091474

ABSTRACT

Parkin is an ubiquitin-protein ligase mutated in Autosomal Recessive - Juvenile Parkinsonism. Here, we describe a cell-based assay to measure Parkin's ubiquitin-protein ligase activity. It relies on the ability of Parkin to recognise depolarised mitochondria and exploits a cell line where Parkin expression is inducible. In these cells, Parkin expression promotes mitophagy and accelerates cell death in response to mitochondrial depolarisers. Time-lapse imaging confirmed cell death and revealed increased perinuclear mitochondrial clustering following induction of Parkin expression in cells exposed to carbonyl cyanide m-chlorophenylhydrazone. Similar effects were not observed with α-synuclein or DJ-1, other proteins associated with the development of Parkinson's disease, confirming the specificity of the assay. We have used this assay to demonstrate that ligase-defective Parkin mutants are inactive, and cellular proteasomal activity (using the proteasomal inhibitors MG132, clasto-lactacystin ß-lactone and epoxomicin) is essential for the Parkin mediated effect. As the assay is suitable for high-throughput screening, it has the potential to identify novel proteostasis compounds that stimulate the activity of Parkin mutants for therapeutic purposes, to identify modulators of kinase activities that impact on Parkin function, and to act as a functional read-out in reverse genetics screens aimed at identifying modifiers of Parkin function during mitophagy.


Subject(s)
Ubiquitin-Protein Ligases/isolation & purification , Ubiquitin-Protein Ligases/metabolism , Blotting, Western/methods , Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Cell Death/drug effects , Cell Death/physiology , HEK293 Cells , Humans , Mitochondria/drug effects , Mitochondria/metabolism , Proteasome Endopeptidase Complex/drug effects , Proteasome Endopeptidase Complex/metabolism , Time-Lapse Imaging/methods , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/biosynthesis , Ubiquitin-Protein Ligases/genetics , Uncoupling Agents/pharmacology , alpha-Synuclein/isolation & purification , alpha-Synuclein/metabolism
3.
Neurobiol Aging ; 31(5): 796-804, 2010 May.
Article in English | MEDLINE | ID: mdl-19097673

ABSTRACT

Despite numerous evidences for neurotoxicity of overexpressed alpha-synuclein, a protective function was suggested for endogenous alpha-synuclein and other members of the synuclein family. This protective role is most important for and evident in presynaptic terminals, where synucleins are normally accumulated. However, mice lacking synucleins display no adverse phenotype. In particular, no significant changes in striatal dopamine metabolism and only subtle deficit of dopaminergic neurons in the substantia nigra were found in juvenile or adult mice. To assess whether aging and synuclein deficiency may have additive detrimental effect on the nigrostriatal system, we studied dopaminergic neurons of the substantia nigra and their striatal synapses in 24-26-month-old alpha-synuclein and gamma-synuclein null mutant mice. Significant approximately 36% reduction of the striatal dopamine was found in aging alpha-synuclein, but not gamma-synuclein null mutant mice when compared to age-matching wild type mice. This was accompanied by the reduction of TH-positive fibers in the striatum and decrease of striatal levels of TH and DAT. However, no progressive loss of TH-positive neurons was revealed in the substantia nigra of synuclein-deficient aging animals. Our results are consistent with a hypothesis that alpha-synuclein is important for normal function and integrity of synapses, and suggest that in the aging nervous system dysfunction of this protein could become a predisposition factor for the development of nigrostriatal pathology.


Subject(s)
Aging/metabolism , Corpus Striatum/metabolism , Dopamine/metabolism , Substantia Nigra/metabolism , Synapses/metabolism , alpha-Synuclein/metabolism , Age Factors , Aging/genetics , Animals , Blotting, Western , Cell Count , Chromatography, High Pressure Liquid , Immunohistochemistry , Mice , Mice, Knockout , Nerve Fibers/metabolism , Neurons/metabolism , Synapses/genetics , Tyrosine 3-Monooxygenase/metabolism , alpha-Synuclein/genetics , gamma-Synuclein/genetics , gamma-Synuclein/metabolism
4.
Protein Pept Lett ; 11(3): 229-37, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15182224

ABSTRACT

Lesions known as Lewy bodies (LBs) and Lewy neurites (LNs) characterise brains of Parkinson's disease (PD) patients. Intracellular aggregation of alpha-synuclein (alpha-syn) appears to play a key role in the generation of LBs and LNs. Such aggregation in the presence of redox metals may initiate Fenton reaction-mediated generation of reactive oxygen species (ROS). ROS thus generated may result in cytotoxic mechanisms such as the induction of DNA single-strand breaks.


Subject(s)
Nerve Tissue Proteins/metabolism , Parkinson Disease/metabolism , Parkinson Disease/pathology , Animals , DNA Damage , Humans , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Reactive Oxygen Species/metabolism , Synucleins , alpha-Synuclein
5.
J Neurochem ; 87(3): 620-30, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14535945

ABSTRACT

Lewy bodies in the brains of patients with Parkinson's disease (PD) contain aggregates of alpha-synuclein (alpha-syn). Missense mutations (A53T or A30P) in the gene encoding alpha-syn are responsible for rare, inherited forms of PD. In this study, we explored the susceptibility of untransfected human dopaminergic BE(2)-M17 neuroblastoma cells, cells transfected with vector only, or cells transfected with wild-type alpha-syn, A30P alpha-syn or A53T alpha-syn to Fe(II)-induced DNA damage in the form of single-strand breaks (SSBs). DNA SSBs were detected following 2-h treatments with various concentrations of Fe(II) (0.01-100.0 microm), using the alkaline single cell-gel electrophoresis ('Comet') assay and quantified by measuring comet tail length (CTL) microm). Fe(II) treatment induced significant increases in CTL in cells transfected with A30P alpha-syn or A53T alpha-syn, even at the lowest concentrations of Fe(II) tested. In comparison, untransfected cells, vector control cells or cells transfected with wild-type alpha-syn exhibited increases in SSBs only when exposed to concentrations of 1.0 microm Fe(II) and above. Even when exposed to higher concentrations (10.0-100.0 microm) of Fe(II), untransfected cells, vector control cells or cells transfected with wild-type alpha-syn were less susceptible to DNA-damage induction than cells transfected with A30P alpha-syn or A53T alpha-syn. Incorporation of DNA-repair inhibitors, hydroxyurea and cytosine arabinoside, enhanced the sensitivity of DNA damage detection. Susceptibility to Fe(II)-induced DNA damage appeared to be dependent on alpha-syn status because cells transfected with wild-type alpha-syn or A53T alpha-syn were equally susceptible to the damaging effects of the mitochondrial respiratory chain inhibitor rotenone. Overall, our data are suggestive of an enhanced susceptibility to the toxic effects of Fe(II) in neuroblastoma cells transfected with mutant alpha-syn associated with inherited forms of PD.


Subject(s)
DNA Damage , Dopamine/metabolism , Ferrous Compounds/toxicity , Nerve Tissue Proteins/genetics , Neuroblastoma/metabolism , Amino Acid Substitution , Cell Line, Tumor , Comet Assay , Cytarabine/pharmacology , DNA/analysis , DNA/drug effects , DNA Repair/drug effects , Electron Transport/drug effects , Humans , Hydroxyurea/pharmacology , Nerve Tissue Proteins/biosynthesis , Neuroblastoma/drug therapy , Rotenone/pharmacology , Synucleins , Transfection , Uncoupling Agents/pharmacology , alpha-Synuclein
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