Accumulation of Ubiquitin and Sequestosome-1 Implicate Protein Damage in Diacetyl-Induced Cytotoxicity

作者：Hubbs Ann F^*; Fluharty Kara L; Edwards Rebekah J; Barnabei Jamie L; Grantham John T; Palmer Scott M; Kelly Francine; Sargent Linda M; Reynolds Steven H; Mercer Robert R; Goravanahally Madhusudan P; Kashon Michael L; Honaker John C; Jackson Mark C; Cumpston Amy M; Goldsmith William T; McKinney Walter; Fedan Jeffrey S; Battelli Lori A; Munro Tiffany; Bucklew Moyers Winnie; McKinstry Kimberly; Schwegler Berry Diane; Friend Sherri; Knepp Alycia K

来源：American Journal Of Pathology, 2016, 186(11): 2887-2908.

DOI：10.1016/j.ajpath.2016.07.018

Inhaled diacetyl vapors are associated with flavorings-related lung disease, a potentially fatal airway disease. The reactive alpha-dicarbonyl group in diacetyl causes protein damage in vitro. Dicarbonyl/L-xylulose reductase (DCXR) metabolizes diacetyl into acetoin, which lacks this alpha-dicarbonyl group. To investigate the hypothesis that flavorings-related lung disease is caused by in vivo protein damage, we correlated diacetyl-induced airway damage in mice with immunofluorescence for markers of protein turnover and autophagy. Western immunoblots identified shifts in ubiquitin pools. Diacetyl inhalation caused dose-dependent increases in bronchial epithelial cells with puncta of both total ubiquitin and K63-ubiquitin, central mediators of protein turnover. This response was greater in Dcxr-knockout mice than in wild-type controls inhaling 200 ppm diacetyl, further implicating the alpha-dicarbonyl group in protein damage. Western immunoblots demonstrated decreased free ubiquitin in airway-enriched fractions. Transmission electron microscopy and colocalization of ubiquitin-positive puncta with lysosomal-associated membrane proteins 1 and 2 and with the multifunctional scaffolding protein sequestosome-1 (SQSTM1/p62) confirmed autophagy. Surprisingly, immunoreactive SQSTM1 also accumulated in the olfactory bulb of the brain. Olfactory bulb SQSTM1 often congregated in activated microglial cells that also contained olfactory marker protein, indicating neuronophagia within the olfactory bulb. This suggests the possibility that SQSTM1 or damaged proteins may be transported from the nose to the brain. Together, these findings strongly implicate widespread protein damage in the etiology of flavorings-related lung disease.

• 出版日期2016-11