ABSTRACT
Glucose residues from N-linked oligosaccharides are removed by glucosidases I and II in the endoplasmic reticulum (ER) or by the alternate endomannosidase pathway in the Golgi apparatus. Our morphological analysis demonstrates that recombinant rat endomannosidase exhibited a cis- and medial-Golgi localization alike the endogenous enzyme and its ER to Golgi transport is COP II mediated. Recombinant endomannosidase undergoes a posttranslational modification, which is not related to N-or O-glycosylation. A shift in molecular mass of recombinant endomannosidase was observed upon phosphatase digestion but not for ER-retained CHO cell endomannosidase. Furthermore, immunoprecipitation of (35)S- and (33)P-labeled endomannosidase expressed in CHO-K1 cells suggests that recombinant endomannosidase undergoes phosphorylation. Substitution of the single cytoplasmic threonine residue of rat endomannosidase by either an alanine or valine residue resulted in the same posttranslational modification alike the wild-type enzyme. The subcellular localization and the in vivo activity of the mutant endomannosidase were not affected. Thus, endomannosidase phosphorylation is occurring in luminal sequences. Modification was prevented when endomannosidase was synthesized using reticulocyte lysates in the presence of canine microsomes. Treatment of cells with brefeldin A blocked the posttranslational modification of endomannosidase, suggesting that phosphorylation is occurring in the Golgi apparatus, the residence of endomannosidase.
Subject(s)
Golgi Apparatus/metabolism , Mannosidases/chemistry , Membrane Proteins/chemistry , Animals , CHO Cells , Cricetinae , Cricetulus , Gene Expression Regulation , Glycosylation , Golgi Apparatus/chemistry , Immunohistochemistry , Lectins/chemistry , Microscopy, Immunoelectron/methods , Phosphorylation , Protein Processing, Post-Translational , Rats , alpha-Glucosidases/chemistryABSTRACT
Influenza A virus is an important human pathogen causing significant morbidity and mortality every year and threatening the human population with epidemics and pandemics. Therefore, it is important to understand the biology of this virus to develop strategies to control its pathogenicity. Here, we demonstrate that influenza A virus inhibits macroautophagy, a cellular process known to be manipulated by diverse pathogens. Influenza A virus infection causes accumulation of autophagosomes by blocking their fusion with lysosomes, and one viral protein, matrix protein 2, is necessary and sufficient for this inhibition of autophagosome degradation. Macroautophagy inhibition by matrix protein 2 compromises survival of influenza virus-infected cells but does not influence viral replication. We propose that influenza A virus, which also encodes proapoptotic proteins, is able to determine the death of its host cell by inducing apoptosis and also by blocking macroautophagy.
Subject(s)
Apoptosis , Autophagy , Influenza A virus/pathogenicity , Lysosomes/metabolism , Phagosomes/metabolism , Viral Matrix Proteins/physiology , Virulence Factors/physiology , Animals , Cell Line , Dogs , Epithelial Cells/virology , Humans , Lysosomes/ultrastructure , Mice , Microscopy, Confocal , Microscopy, Electron, Transmission , Phagosomes/ultrastructureABSTRACT
In cells the quality of newly synthesized proteins is monitored in regard to proper folding and correct assembly in the early secretory pathway, the cytosol and the nucleoplasm. Proteins recognized as non-native in the ER will be removed and degraded by a process termed ERAD. ERAD of aberrant proteins is accompanied by various changes of cellular organelles and results in protein folding diseases. This review focuses on how the immunocytochemical labeling and electron microscopic analyses have helped to disclose the in situ subcellular distribution pattern of some of the key machinery proteins of the cellular protein quality control, the organelle changes due to the presence of misfolded proteins, and the efficiency of synthetic chaperones to rescue disease-causing trafficking defects of aberrant proteins.
Subject(s)
Endoplasmic Reticulum/metabolism , Molecular Chaperones/metabolism , Protein Folding , Proteins/metabolism , Drug Design , Endoplasmic Reticulum/ultrastructure , Humans , Membrane Proteins/physiology , Metabolism, Inborn Errors/drug therapy , Metabolism, Inborn Errors/etiology , Molecular Chaperones/therapeutic use , Proteins/geneticsABSTRACT
Cystic fibrosis (CF) is caused by mutations of the gene encoding for the CFTR (CF transmembrane conductance regulator) protein. The most frequent mutation, the (Delta)F508 mutation, results in a defective cAMP-regulated chloride transport in the epithelial cells. The spectrum of clinical manifestations in patients bearing homozygous (Delta)F508 mutations can vary considerably, suggesting that, in the patients with a mild disease, CFTR could be partly functional. To test this hypothesis, we explored in nasal ciliated epithelial cells (NCC) of 9 control subjects and 23 (Delta)F508 homozygous patients the anion conductive pathway by a halide sensitive fluorescent dye assay SPQ (6-methoxy-N-3'-sulfopropylquinolinium) and the CFTR transcript levels by RT-PCR. As 50% represented the lowest fraction of the control subjects NCC demonstrating a cAMP-dependent conductance, a CF patient was considered as "cAMP responder" if at least 50% of the NCC tested displayed a cAMP-dependent conductive pathway. According to these criteria, 8 of the 23 patients were considered as cAMP responders. They had a significantly less severe disease considering the respiratory function and infectious status. The amount of CFTR mRNA did not differ between the control subjects and the patients. No statistical correlation could be found between the transcript level and the expression of a cAMP conductive pathway. This cAMP-dependent Cl(-) conductance detected in homozygous NCC could be due to a residual CFTR activity and may explain the mild phenotypes observed in some (Delta)F508 homozygous patients.
