engleski

Cigarette smoke - induced protein damage: role of the proteasome

Introduction: The mechanism by which cigarette smoking leads to the development of chronic obstructive pulmonary disease is still unclear. Cigarette smoke contributes to the oxidative damage of proteins inside the lung. There are two main systems to protect eucariotic cells from unfolded and damaged proteins: heat shock proteins and the ubiquitin-proteasome pathway. Any stressful condition that causes damage to cell proteins can induce heat shock protein synthesis. Degradation of proteins via the ubiquitin-proteasome pathway involves conjugation of multiple ubiquitin moieties and degradation of the tagged protein by the proteasome into small peptides. We have tested if the proteasome is involved in the increase of amino acid content inside the gas phase cigarette smoke-treated airway epithelial cells. Furthermore, we hypothesize that cigarette smoke causes protein damage, induces HSP70 expression and increases amount of ubiquitin-protein conjugates in epithelial cells. We have also examined weather cigarette smoke disturbs proteasomal degradation of damaged proteins. Materials and methods: A549 cells were exposed to gas phase cigarette smoke (GPCS) or cigarette smoke extract (CSE) for 4 or 24 hours. Free amino acids from GPCS-treated A549 cell lysates were quantified by Liquid Chromatography - Mass Spectrometry using stable isotope labeling. HSP70 and ubiquitin - protein conjugates were analyzed by Western blot. Proteasomal subunits were labeled by using the fluorescent activity-based probe MV151 and visualised by sensitive in-gel detection method. Proteasomal activities were determined by using fluorogenic substrates. Results: GPCS and CSE cause an increase of ubiquitin - protein conjugates in A549 cells. The proteasome is involved in the increase of free amino acids inside GPCS-treated A549 cells (p < 0.05). CSE causes an increase of HSP70 expression (biological duplicate). Proteasomal active sites were not disturbed by GPCS or CSE, except after 24-hour incubation of A549 cells stimulated with GPCS. GPCS changes all three kinds of activities of the proteasome. Conclusions: Our data suggest that cigarette smoke induces damage to proteins which are subsequently ubiquitinated and tagged for proteasomal degradation. Both proteasome and HSP70 are involved in the elimination of proteins damaged by cigarette smoke. Although proteasomal active sites remain intact, cigarette smoke significantly disturbs proteasomal activity which may contribute to the aggregation of damaged proteins and enhanced cell death.

cigarette smoking ; cronic obstructive pulmonary disease ; ubiquitin-proteasomal pathway ; HSP70

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