Cigarette smoke exposure enhances transforming acidic coiled-coil–containing protein 2 turnover and thereby promotes emphysema
Rama K. Mallampalli, Xiuying Li, Jun-Ho Jang, Tomasz Kaminski, Aki Hoji, Tiffany Coon, Divay Chandra, Starr Welty, Yaqun Teng, John Sembrat, Mauricio Rojas, Yutong Zhao, Robert Lafyatis, Chunbin Zou, Frank Sciurba, Prithu Sundd, Li Lan, Toru Nyunoya
Rama K. Mallampalli, Xiuying Li, Jun-Ho Jang, Tomasz Kaminski, Aki Hoji, Tiffany Coon, Divay Chandra, Starr Welty, Yaqun Teng, John Sembrat, Mauricio Rojas, Yutong Zhao, Robert Lafyatis, Chunbin Zou, Frank Sciurba, Prithu Sundd, Li Lan, Toru Nyunoya
View: Text | PDF
Research Article Cell biology Pulmonology

Cigarette smoke exposure enhances transforming acidic coiled-coil–containing protein 2 turnover and thereby promotes emphysema

Abstract

Our integrative genomic and functional analysis identified transforming acidic coiled-coil–containing protein 2 (TACC2) as a chronic obstructive pulmonary disease (COPD) candidate gene. Here, we found that smokers with COPD exhibit a marked decrease in lung TACC2 protein levels relative to smokers without COPD. Single cell RNA sequencing reveals that TACC2 is expressed primarily in lung epithelial cells in normal human lungs. Furthermore, suppression of TACC2 expression impairs the efficiency of homologous recombination repair and augments spontaneous and cigarette smoke extract–induced (CSE-induced) DNA damage and cytotoxicity in immortalized human bronchial epithelial cells. By contrast, enforced expression of TACC2 attenuates the CSE effects. We also found that CSE enhances TACC2 degradation via the ubiquitin-proteasome system mediated by the ubiquitin E3 ligase subunit, F box L7. Furthermore, cellularly expressed TACC2 proteins harboring naturally occurring mutations exhibited altered protein lifespan coupled with modified DNA damage repair and cytotoxic responses. CS triggers emphysematous changes accompanied by accumulated DNA damage, apoptosis of alveolar epithelia, and lung inflammation in Tacc2–/– compared with Tacc2+/+ mice. Our results suggest that CS destabilizes TACC2 protein in lung epithelia by the ubiquitin proteasome system, leading to subsequent DNA damage, cytotoxicity, and emphysema.

Authors

Rama K. Mallampalli, Xiuying Li, Jun-Ho Jang, Tomasz Kaminski, Aki Hoji, Tiffany Coon, Divay Chandra, Starr Welty, Yaqun Teng, John Sembrat, Mauricio Rojas, Yutong Zhao, Robert Lafyatis, Chunbin Zou, Frank Sciurba, Prithu Sundd, Li Lan, Toru Nyunoya

×

Figure 5

An SCF E3 Ligase F-box component targets TACC2 for polyubiquitination.

Options: View larger image (or click on image) Download as PowerPoint
An SCF E3 Ligase F-box component targets TACC2 for polyubiquitination. (...
(A) HBEC2 cells were cultured with 2% CSE and 20 μg/mL CHX in the presence or absence of 10 μM MG132 or 10 μM leupeptin for up to 8 hours. IB analysis was performed for TACC2. IB data are representative of 3 independent experiments. (B) BEAS-2B cells were transfected with the HA-ubiquitin plasmid (1.5 or 3.0 μg per well in a 6-well plate). Cell lysates were processed for TACC2 IB (above). TACC2 was also immunoprecipitated (IP) and samples processed for HA IB (middle panel). IB data are representative of 3 independent experiments. (C) BEAS-2B cells were cultured without or with 2% CSE in the presence of 10 μM MG132 for 2 hours. IP was performed with primary TACC2 antibody using cell lysates. IB analysis was performed for levels of ubiquitination (Ub), TACC2, and GAPDH. IB data are representative of 3 independent experiments. (D) BEAS-2B cells were transfected using nucleofection with increasing amounts of V5-tagged FBXL7 or FBXL8-encoding plasmids for 48 hours. IB data of TACC2 and F-box proteins are representative of 3 independent experiments. (E) BEAS-2B cells were cultured without or with 2% CSE for 4 hours. IP was performed with primary TACC2 antibody using cell lysates. IB analysis was performed for TACC2, FBXL7, and GAPDH. IB data are representative of 3 independent experiments. (F) In vitro ubiquitination of TACC2. Recombinant ubiquitin E1–activating, -E2–conjugating enzymes and ubiquitin was incubated without or with FBXL7 in the presence of V5-TACC2, and polyubiquitinylated products were detected using V5 IB. (G) BEAS-2B cells were transfected with V5-tagged TACC2 WT and phosphorylation-defective mutant (T151A, S304A, S395A, and S399A) plasmids for 48 hours and further incubated with 2% CSE for 4 hours. IP was performed using primary V5 antibody in cell lysates. IB analysis was performed using V5, GAPDH, and FBXL7 antibodies.