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Cell Biology and Toxicology

, Volume 35, Issue 4, pp 345–360 | Cite as

Protein carbonylation in human bronchial epithelial cells exposed to cigarette smoke extract

  • Graziano ColomboEmail author
  • Maria Lisa Garavaglia
  • Emanuela Astori
  • Daniela Giustarini
  • Ranieri Rossi
  • Aldo Milzani
  • Isabella Dalle-Donne
Original Article

Abstract

Cigarette smoke is a well-established exogenous risk factor containing toxic reactive molecules able to induce oxidative stress, which in turn contributes to smoking-related diseases, including cardiovascular, pulmonary, and oral cavity diseases. We investigated the effects of cigarette smoke extract on human bronchial epithelial cells. Cells were exposed to various concentrations (2.5–5–10–20%) of cigarette smoke extract for 1, 3, and 24 h. Carbonylation was assessed by 2,4-dinitrophenylhydrazine using both immunocytochemical and Western immunoblotting assays. Cigarette smoke induced increasing protein carbonylation in a concentration-dependent manner. The main carbonylated proteins were identified by means of two-dimensional electrophoresis coupled to MALDI-TOF mass spectrometry analysis and database search (redox proteomics). We demonstrated that exposure of bronchial cells to cigarette smoke extract induces carbonylation of a large number of proteins distributed throughout the cell. Proteins undergoing carbonylation are involved in primary metabolic processes, such as protein and lipid metabolism and metabolite and energy production as well as in fundamental cellular processes, such as cell cycle and chromosome segregation, thus confirming that reactive carbonyl species contained in cigarette smoke markedly alter cell homeostasis and functions.

Keywords

Cigarette smoke extract (CSE) 2D-electrophoresis Human bronchial epithelial cells (16-HBE cells) Matrix-assisted laser desorption/ionization (MALDI) Protein carbonylation 

Abbreviations

2D-GE

2D-gel electrophoresis

Dinitrophenyl-KLH

Keyhole limpet haemocyanin conjugate

DNPH

Dinitrophenylhydrazine

DTT

Dithiothreitol

ECL

Enhanced chemiluminescence

HMW

High molecular weight

HRP

Horseradish peroxidase

MALDI-TOF

Matrix-assisted laser desorption/ionization time of flight

MS

Mass spectrometry

Notes

Acknowledgments

This research was supported by the “Piano di Sostegno alla Ricerca 2016—Linea 2” (Università degli Studi di Milano).

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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Graziano Colombo
    • 1
    Email author
  • Maria Lisa Garavaglia
    • 1
  • Emanuela Astori
    • 1
  • Daniela Giustarini
    • 2
  • Ranieri Rossi
    • 2
  • Aldo Milzani
    • 1
  • Isabella Dalle-Donne
    • 1
  1. 1.Department of BiosciencesUniversità degli Studi di MilanoMilanItaly
  2. 2.Department of Biotechnology, Chemistry and PharmacyUniversity of SienaSienaItaly

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