Sepsis due to infection with highly virulent bacteria, such as P. aeruginosa, is the most common cause of the acute respiratory distress syndrome (ARDS). P. aeruginosa causes lung injury by greatly decreasing synthesis and availability of phosphatidylcholine (PC), the major surfactant phospholipid. This proposal will investigate how P. aeruginosa modulates surfactant production by altering function of the key enzyme, cytidylyltransferase (CCT). The PI has uncovered several novel preliminary observations: (i) P. aeruginosa rapidly triggers CCT nuclear import in lung epithelia, a process involving CCT interaction with Ca++/CaM/CaM kinase I, and 14-3-3?, the latter which regulates protein trafficking, (ii) P. aeruginosa potently degrades CCT protein via a Ca++-activated E3-ligase mediated monoubiquitin-dependent degradation mechanism, and (iii) that CaM protects CCT from E3-ligase ubiquitin degradation. Thus, alveolar epithelia harbor two intrinsic homeostatic control mechanisms to maintain surfactant PC synthesis after bacterial infection. The first mechanism involves CCT nuclear entry as an acute salvage response;the second mechanism is protection of CCT proteolysis by CaM. When these pathways are overwhelmed by bacterial infection, CCT is degraded by ubiquitin-dependent processing. These results led to the overall hypothesis that calcium-CaM interactions play a central regulatory role in salvage and degradative pathways for the surfactant enzyme, CCT, in response to bacterial infection in lung epithelia. The PI will determine if 14-3-3? is a molecular chaperone that escorts CCT for nuclear import in vitro and in vivo in a Ca++/CaM/CaM kinase 1-regulated manner. This mechanism may serve as an initial salvage pathway after P. aeruginosa infection to preserve surfactant synthesis (Aim 1). Second, the PI will determine if the E3 ligase, FBL2, triggers ubiquitin-dependent degradation of CCT in a Ca++/CaM-regulated manner after long-term P. aeruginosa infection, an effect antagonized by CaM (Aim 2). The PI will use molecular and biochemical approaches to identify the molecular signatures that direct interactions between these CCT regulatory proteins and will determine their functional significance in murine models of bacterial infection. Studies will entail expression of novel E3 ligase resistant CCT enzyme mutants that exhibit robust catalytic activity and yet are less sensitive to proteolytic modification after P. aeruginosa-induced acute lung injury. These studies lay the foundation for generating small molecule (i.e. drug) CCT or 14-3-3 activators or specific E3 ligase inhibitors for use in surfactant-deficient states. Execution of these studies will lay the groundwork for a significant mechanistic advance with regard to the pathobiology of surfactant metabolism and mechanisms for alveolar homeostasis during inflammatory lung injury.

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 protect a key surfactant synthetic enzyme, CCT: i) CCT shifts to the nucleus of lung epithelia by binding to a nuclear trafficking protein, termed 14-3-3, and ii) CCT is stabilized from its breakdown by calmodulin. After severe infection, these mechanisms are exhausted and CCT is rapidly degraded. In this application we will use several tools to confirm that 14-3-3 and calmodulin are indispensable for CCT to maintain sufficient surfactant production and uncover how CCT is degraded.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL097376-04
Application #
8292085
Study Section
Special Emphasis Panel (ZRG1-RES-B (03))
Program Officer
Harabin, Andrea L
Project Start
2009-09-30
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2014-06-30
Support Year
4
Fiscal Year
2012
Total Cost
$393,784
Indirect Cost
$129,811
Name
University of Pittsburgh
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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