benzyloxycarbonylleucyl-leucyl-leucine-aldehyde and lysyl-aspartyl-glutamyl-leucine

Clusterin is a usually secreted glycoprotein with chaperone properties. Recently, it has been suggested that clusterin isoforms reside in the nuclear and cytosolic compartments of human cell types, where they can influence various cellular programs including DNA repair, transcription and apoptosis. Several mechanisms have been proposed to explain this atypical location, including alternative transcription initiation and alternative splicing. However, none of these have been unequivocally established as occurring in live cells. Here we provide direct experimental evidence that in live intact cells, under certain stress conditions, clusterin can evade the secretion pathway and reach the cytosol. This was demonstrated using several complementary approaches. Flow cytometry and selective permeabilization of U251 cell membranes with digitonin allowed detection of cytosolic clusterin in stressed U251 cells. In addition, a stringent enzymatic assay reliant upon the exclusively cytosolic deubiquitinase enzymes confirmed that clusterin synthesized with its hydrophobic secretion signal sequence can reach the cytosol of U251 cells. The retrotranslocation of clusterin is likely to occur through a mechanism similar to the endoplasmic reticulum (ER)-associated protein degradation pathway and involves passage through the Golgi apparatus. We also report that the ER-associated ubiquitin ligase Hrd1/synoviolin can interact with, and ubiquitinate clusterin. The possible biological functions of these novel behaviours of clusterin are discussed.

Topics: Animals; Brefeldin A; Cell Line, Tumor; Chelating Agents; Chlorocebus aethiops; Clusterin; COS Cells; Cysteine Proteinase Inhibitors; Cytosol; Egtazic Acid; Endopeptidases; Endoplasmic Reticulum; Flow Cytometry; Golgi Apparatus; Green Fluorescent Proteins; Humans; Leupeptins; Microscopy, Fluorescence; Oligopeptides; Potassium Chloride; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Sorting Signals; Protein Transport; Recombinant Fusion Proteins; Transfection; Ubiquitin

Mutations in the gene encoding SP-C (surfactant protein C; SFTPC) have been linked to interstitial lung disease (ILD) in children and adults. Expression of the index mutation, SP-C(Deltaexon4), in transiently transfected cells and type II cells of transgenic mice resulted in misfolding of the proprotein, activation of endoplasmic reticulum (ER) stress pathways, and cytotoxicity. In this study, we show that stably transfected cells adapted to chronic ER stress imposed by the constitutive expression of SP-C(Deltaexon4) via an NF-kappaB-dependent pathway. However, the infection of cells expressing SP-C(Deltaexon4) with respiratory syncytial virus resulted in significantly enhanced cytotoxicity associated with accumulation of the mutant proprotein, pronounced activation of the unfolded protein response, and cell death. Adaptation to chronic ER stress imposed by misfolded SP-C was associated with increased susceptibility to viral-induced cell death. The wide variability in the age of onset of ILD in patients with SFTPC mutations may be related to environmental insults that ultimately overwhelm the homeostatic cytoprotective response.

Topics: Animals; Apoptosis; Caspase 3; Caspases; Cell Line; Cell Survival; DNA-Binding Proteins; Down-Regulation; Epithelial Cells; Gene Expression; Gene Expression Profiling; Genetic Predisposition to Disease; Humans; I-kappa B Proteins; Immunohistochemistry; Leupeptins; Lung Diseases, Interstitial; Lysosomal Membrane Proteins; Mice; Mice, Transgenic; Mutation; NF-kappa B p50 Subunit; NF-KappaB Inhibitor alpha; Nuclear Proteins; Oligopeptides; Peptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Folding; Protein Sorting Signals; Pulmonary Surfactant-Associated Protein C; Regulatory Factor X Transcription Factors; Respiratory Syncytial Viruses; Transcription Factors; Transfection; Up-Regulation