engleski

Cigarette smoke disturbs protein turnover inside airway epithelial cells

Background: Cigarette smoking causes oxidative stress and severe damage of proteins in the lungs. One of the main systems to protect cells from damaged proteins is the ubiquitin-proteasome pathway. In this work, we follow up on an earlier observation that cigarette smoke (CS)-treated airway epithelial cells contain increased levels of free intracellular amino acids (AA). Objective: We hypothesize that i) CS-induced increased levels of free intracellular AA are due to activation of the proteasome, ii) CS increases the amount of ubiquitin-protein conjugates, iii) CS interferes with proteasomal degradation and de novo protein synthesis. Materials and methods: Airway epithelial cells (A549) were exposed to CS for 1 or 5 min and cultured for the next 4 or 24 h. Ubiquitin-protein conjugates were analyzed by Western blot. Proteasomal subunits were analyzed using the fluorescent activity-based probe MV151. Proteasomal activities were determined using fluorogenic substrates. Free amino acids in the lysates of CS-treated cells were quantified by Liquid Chromatography - Mass Spectrometry using stable isotope labeling. De novo protein synthesis was assayed using the AA analog L-azidohomoalanine. Results: Exposure of airway epithelial cells to CS increased the amount of ubiquitin-protein conjugates. Degradation of ubiquitin-protein conjugates by the proteasome was accompanied by an increase in free intracellular AA and inhibition of nascent protein synthesis. Activity-based labeling of the proteasomal active sites was not affected, except after 5-min exposure to CS. Caspase- and chymotrypsin-like proteasomal activities decreased, while trypsin-like activity increased. Conclusions: Our data imply that CS induces damage to proteins which are subsequently ubiquitinated and directed for proteasomal degradation. Proteasomal activities and protein synthesis are disturbed depending on the exposure time to CS. Inadequate protein turnover due to the effect of CS may thus be related to the disruption of normal structure and functions of the airway epithelium as observed in smokers.

cigarette smoke ; ubiquitin-protein conjugates ; proteasome ; amino acids ; protein synthesis

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