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Dissecting the Molecular Effects of Cigarette Smoke on Proteasome Function
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A7606 - Dissecting the Molecular Effects of Cigarette Smoke on Proteasome Function
Author Block: S. Meiners1, I. E. Kammerl1, A. M. Caniard2, J. Merl-Pham3, G. Ben-Nissan4, A. Mossina1, A. Geerlof3, O. Eickelberg5, S. Hauck3, M. Sharon4; 1CPC, Munich, Germany, 2CPC, Munchen, Germany, 3Helmholtz Zentrum Muenchen, Munich, Germany, 4Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, Israel, 5Pulmonary Sciences and Critical Care Medicine, Univ of Colorado Denver, Denver, CO, United States.
Rationale:
The proteasome plays an essential role for cellular function in controlling the half-life of more than 80% of all cellular proteins. It also represents a major protein quality control system by degrading (oxidatively) damaged proteins. Proteasome dysfunction is emerging as a novel pathomechanism for the development of chronic obstructive pulmonary disease (COPD), a major leading cause of death in the world. Cigarette smoke is one of the main risk factors for COPD and has been shown to impair proteasome function in vitro and in vivo. We have previously shown that proteasome activity is inhibited in COPD lungs while expression levels of proteasome subunits were not altered (Kammerl et al. AJRCCM 2016).
Methods and Results:
In the present study, we dissected the molecular changes induced by cigarette smoke on proteasome function in lung epithelial cells and mouse lungs. We analyzed the integrity, composition, and interactome of 26S proteasome complexes isolated by affinity-purification from cigarette smoke extract (CSE)-exposed A549 cells (24 h exposure) and lungs from C57BL/6 mice exposed to cigarette smoke for three days. Moreover, we investigated whether reactive compounds of cigarette smoke directly modify and inhibit the 20S proteasome complex by applying native MS analysis of 20S proteasome complexes isolated from HEK293 cells that had been acutely exposed to CSE for 30 minutes.
Our data reveal that the 20S proteasome is slightly destabilized in the absence of any dominant modification of proteasomal proteins. 26S pull-down and stoichiometry analysis indicated that 26S proteasome complexes become instable in response to cigarette smoke exposure. Of note, the interactome of the 26S proteasome was clearly altered in smoke-exposed mouse lungs possibly reflecting an altered cellular composition of the lung in response to cigarette smoke exposure.
Conclusion:
Taken together, our results suggest that cigarette smoke induces minor but detectable changes in the stability and interactome of 20S and 26S proteasome complexes, which in a chronic setting might contribute to imbalanced proteostasis as observed in smoke-related chronic lung diseases.
Author Block: S. Meiners1, I. E. Kammerl1, A. M. Caniard2, J. Merl-Pham3, G. Ben-Nissan4, A. Mossina1, A. Geerlof3, O. Eickelberg5, S. Hauck3, M. Sharon4; 1CPC, Munich, Germany, 2CPC, Munchen, Germany, 3Helmholtz Zentrum Muenchen, Munich, Germany, 4Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, Israel, 5Pulmonary Sciences and Critical Care Medicine, Univ of Colorado Denver, Denver, CO, United States.
Rationale:
The proteasome plays an essential role for cellular function in controlling the half-life of more than 80% of all cellular proteins. It also represents a major protein quality control system by degrading (oxidatively) damaged proteins. Proteasome dysfunction is emerging as a novel pathomechanism for the development of chronic obstructive pulmonary disease (COPD), a major leading cause of death in the world. Cigarette smoke is one of the main risk factors for COPD and has been shown to impair proteasome function in vitro and in vivo. We have previously shown that proteasome activity is inhibited in COPD lungs while expression levels of proteasome subunits were not altered (Kammerl et al. AJRCCM 2016).
Methods and Results:
In the present study, we dissected the molecular changes induced by cigarette smoke on proteasome function in lung epithelial cells and mouse lungs. We analyzed the integrity, composition, and interactome of 26S proteasome complexes isolated by affinity-purification from cigarette smoke extract (CSE)-exposed A549 cells (24 h exposure) and lungs from C57BL/6 mice exposed to cigarette smoke for three days. Moreover, we investigated whether reactive compounds of cigarette smoke directly modify and inhibit the 20S proteasome complex by applying native MS analysis of 20S proteasome complexes isolated from HEK293 cells that had been acutely exposed to CSE for 30 minutes.
Our data reveal that the 20S proteasome is slightly destabilized in the absence of any dominant modification of proteasomal proteins. 26S pull-down and stoichiometry analysis indicated that 26S proteasome complexes become instable in response to cigarette smoke exposure. Of note, the interactome of the 26S proteasome was clearly altered in smoke-exposed mouse lungs possibly reflecting an altered cellular composition of the lung in response to cigarette smoke exposure.
Conclusion:
Taken together, our results suggest that cigarette smoke induces minor but detectable changes in the stability and interactome of 20S and 26S proteasome complexes, which in a chronic setting might contribute to imbalanced proteostasis as observed in smoke-related chronic lung diseases.
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