Sepsis-induced acute lung injury is associated with greatly decreased synthesis of surfactant, a stabilizing material comprised mainly of the lipid, phosphatidylcholine (PC). The production of PC is exquisitely controlled by the key surfactant enzyme, CCT. CCT is prone to phosphorylation and proteolytic degradation leading to enzyme inactivation and reduced surfactant availability. Our preliminary data show that in bacterial sepsis models, intracellular calcium triggers kinase-mediated CCT phosphorylation (Aim 1) and F-Box driven ubiquitination and lysosomal degradation of the surfactant enzyme (Aim 2) that impairs surfactant production. Calmodulin, a calcium-regulated sensor appears to protect CCT from these modifications. Thus, we hypothesize that in sepsis, lung injury is mediated, in part, by post-translational events within the CCT primary structure. We will test our hypothesis using state-of-art approaches to identify putative phosphorylation and ubiquitin acceptor sites within the CCT sequence and molecular signatures that govern interactions between F-Box, calmodulin, ubiquitin, and the CCT phosphorylation state. We will also examine the ability of calmodulin to oppose action of F-Box and kinase activity in vitro and in vivo. Our approaches include adenoviral gene transfer of novel surfactant CCT enzymes that are resistant to these post-translational modifications and calmodulin gene transfer in mice after bacterial sepsis. Our long-term goal is to devise suitable pharmaceutical inhibitors of bacterially regulated F-box proteins and kinases or calmodulin activators that might stimulate surfactant production in florid sepsis.

Public Health Relevance

Sepsis-induced acute lung injury results in decreased production of surfactant, an essential material that stabilizes lung function. We have discovered that in animal models of septic lung injury, there are two mechanisms that greatly reduce function of a key surfactant synthetic enzyme, CCT: i) CCT phosphorylation by stress kinase, and ii) CCT is degraded a F-Box E 3 ligase;these effects are opposed by calmodulin. In this application we will use several tools to confirm that stress kinase and F-Box are critical factors that reduce CCT availability and surfactant production after bacterial infection. Execution of studies outlined in this application will provide a major advance in the conceptual framework for how sepsis causes lung impairment by altering surfactant production.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL098174-05
Application #
8602852
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Harabin, Andrea L
Project Start
2010-02-01
Project End
2015-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
5
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Lendermon, Elizabeth A; Coon, Tiffany A; Bednash, Joseph S et al. (2017) Azithromycin decreases NALP3 mRNA stability in monocytes to limit inflammasome-dependent inflammation. Respir Res 18:131
Suber, Tomeka; Wei, Jianxin; Jacko, Anastasia M et al. (2017) SCFFBXO17 E3 ligase modulates inflammation by regulating proteasomal degradation of glycogen synthase kinase-3? in lung epithelia. J Biol Chem 292:7452-7461
Bednash, Joseph S; Weathington, Nathaniel; Londino, James et al. (2017) Targeting the deubiquitinase STAMBP inhibits NALP7 inflammasome activity. Nat Commun 8:15203
Londino, James D; Gulick, Dexter L; Lear, Travis B et al. (2017) Post-translational modification of the interferon-gamma receptor alters its stability and signaling. Biochem J 474:3543-3557
Evankovich, John; Lear, Travis; Mckelvey, Alison et al. (2017) Receptor for advanced glycation end products is targeted by FBXO10 for ubiquitination and degradation. FASEB J 31:3894-3903
Han, SeungHye; Jerome, Jacob A; Gregory, Alyssa D et al. (2017) Cigarette smoke destabilizes NLRP3 protein by promoting its ubiquitination. Respir Res 18:2
Liu, Yuan; Mallampalli, Rama K (2016) Small molecule therapeutics targeting F-box proteins in cancer. Semin Cancer Biol 36:105-19
Zou, Chunbin; Synan, Matthew J; Li, Jin et al. (2016) LPS impairs oxygen utilization in epithelia by triggering degradation of the mitochondrial enzyme Alcat1. J Cell Sci 129:51-64
Bednash, Joseph S; Mallampalli, Rama K (2016) Regulation of inflammasomes by ubiquitination. Cell Mol Immunol 13:722-728
Krzysiak, Troy C; Chen, Bill B; Lear, Travis et al. (2016) Crystal structure and interaction studies of the human FBxo3 ApaG domain. FEBS J 283:2091-101

Showing the most recent 10 out of 57 publications