Bik promotes proteasomal degradation to control low-grade inflammation
Yohannes A. Mebratu, Jane T. Jones, Congjian Liu, Zerihun H. Negasi, Mizanur Rahman, Joselyn Rojas-Quintero, George T. O’Connor, Wei Gao, Josée Dupuis, Michael H. Cho, Augusto A. Litonjua, Scott Randell, Yohannes Tesfaigzi
Yohannes A. Mebratu, Jane T. Jones, Congjian Liu, Zerihun H. Negasi, Mizanur Rahman, Joselyn Rojas-Quintero, George T. O’Connor, Wei Gao, Josée Dupuis, Michael H. Cho, Augusto A. Litonjua, Scott Randell, Yohannes Tesfaigzi
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Research Article Inflammation

Bik promotes proteasomal degradation to control low-grade inflammation

Abstract

Although chronic low-grade inflammation does not cause immediate clinical symptoms, over the longer term, it can enhance other insults or age-dependent damage to organ systems and thereby contribute to age-related disorders, such as respiratory disorders, heart disease, metabolic disorders, autoimmunity, and cancer. However, the molecular mechanisms governing low-level inflammation are largely unknown. We discovered that Bcl-2–interacting killer (Bik) deficiency causes low-level inflammation even at baseline and the development of spontaneous emphysema in female but not male mice. Similarly, a single nucleotide polymorphism that reduced Bik levels was associated with increased inflammation and enhanced decline in lung function in humans. Transgenic expression of Bik in the airways of Bik-deficient mice inhibited allergen- or LPS-induced lung inflammation and reversed emphysema in female mice. Bik deficiency increased nuclear but not cytosolic p65 levels because Bik, by modifying the BH4 domain of Bcl-2, interacted with regulatory particle non-ATPase 1 (RPN1) and RPN2 and enhanced proteasomal degradation of nuclear proteins. Bik deficiency increased inflammation primarily in females because Bcl-2 and Bik levels were reduced in lung tissues and airway cells of female compared with male mice. Therefore, controlling low-grade inflammation by modifying the unappreciated role of Bik and Bcl-2 in facilitating proteasomal degradation of nuclear proteins may be crucial in treating chronic age-related diseases.

Authors

Yohannes A. Mebratu, Jane T. Jones, Congjian Liu, Zerihun H. Negasi, Mizanur Rahman, Joselyn Rojas-Quintero, George T. O’Connor, Wei Gao, Josée Dupuis, Michael H. Cho, Augusto A. Litonjua, Scott Randell, Yohannes Tesfaigzi

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Figure 4

The BH3 domain of Bik inhibits nuclear p65–induced transcriptional activity.

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The BH3 domain of Bik inhibits nuclear p65–induced transcriptional activ...
A549 NF-κB luciferase reporter cells were infected with either Ad GFP, Ad-Bik, or Ad-BikL61G (mutant Bik that does not kill cells) and subsequently treated with 10 ng/ml TNF-α for 6 hours. (A) Protein lysates were analyzed for the expression of Bik by Western blotting. (B) Percentages of viable cells were analyzed by trypan blue exclusion assay. n = 3. Cells were infected with the adenoviral vectors at MOIs indicated below the bar. (C) NF-κB transcriptional activity was analyzed in the cell lysates. n = 4. (D) Percentages of cells expressing nuclear p65 were analyzed by immunofluorescent staining. n = 6–8; N = 2. (E) A549 NF-κB luciferase reporter cells were treated with vehicle, TAT, or TAT-BikL61G peptides for 2 hours and subsequently treated with TNF-α for 6 hours. NF-κB transcriptional activity was analyzed in the cell lysates. n = 3. (F) A549 cells were treated with 10 μM control peptides or BikL61G peptides for 2 hours, followed by treatments with 10 ng/ml TNF-α for the indicated times in the presence of phosphormide. Cell lysates were analyzed for the level of IκBα protein by Western blotting. (G) Cytosolic-protein lysates from bik+/+ and bik–/– MAECs were analyzed for levels of phospho- and total IκBα by Western blotting. (H) Cytosolic lysates from bik+/+ and bik–/– MAECs were immunoprecipitated using anti-p65 antibody and analyzed for IκBα levels by Western blotting. (I) Cytosolic and nuclear fractions of lysates from bik+/+ and bik–/– MAECs were analyzed for levels of p65 and IκBα by Western blotting. (J) Nuclear fractions of lysates isolated from bik+/+ and bik–/– MAECs analyzed for levels of p65 and p50 by Western blotting. (K) HAECs transfected with siControl or IRF-1 siRNA. Western blot of nuclear lysates for Bik and p65 protein. (L) bik–/– mice instilled with 50 μg HDM intranasally daily for 5 consecutive days and on days 6 and 7 intranasally treated with 10 μM of control TAT peptide, BH3 WT Bik peptide, or BH3 mutant Bik peptide. BAL fluids analyzed for inflammatory cell numbers. n = 4–6/group. Two-tailed Student’s t test was used to compare between 2 groups, and grouped results were analyzed using 2-way ANOVA. Data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.