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.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-RES-B (03))
Program Officer
Harabin, Andrea L
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Pittsburgh
Internal Medicine/Medicine
Schools of Medicine
United States
Zip Code
Chen, Yan; Li, Jin; Dunn, Sarah et al. (2014) Histone deacetylase 2 (HDAC2) protein-dependent deacetylation of mortality factor 4-like 1 (MORF4L1) protein enhances its homodimerization. J Biol Chem 289:7092-8
Goetzman, Eric S; Alcorn, John F; Bharathi, Sivakama S et al. (2014) Long-chain acyl-CoA dehydrogenase deficiency as a cause of pulmonary surfactant dysfunction. J Biol Chem 289:10668-79
Zou, Chunbin; Mallampalli, Rama K (2014) Regulation of histone modifying enzymes by the ubiquitin-proteasome system. Biochim Biophys Acta 1843:694-702
Weathington, Nathaniel M; Snavely, Courtney A; Chen, Bill B et al. (2014) Glycogen synthase kinase-3? stabilizes the interleukin (IL)-22 receptor from proteasomal degradation in murine lung epithelia. J Biol Chem 289:17610-9
Chen, Bill B; Coon, Tiffany A; Glasser, Jennifer R et al. (2014) E3 ligase subunit Fbxo15 and PINK1 kinase regulate cardiolipin synthase 1 stability and mitochondrial function in pneumonia. Cell Rep 7:476-87
Han, SeungHye; Mallampalli, Rama K (2014) Sizing up surfactant synthesis. Cell Metab 20:195-6
Weathington, Nathaniel M; Mallampalli, Rama K (2014) Emerging therapies targeting the ubiquitin proteasome system in cancer. J Clin Invest 124:6-12
Liu, Yuan; Mallampalli, Rama K (2014) Decoding the growth advantage of hypoxia-sensitive lung cancer. Am J Respir Crit Care Med 190:603-5
Agassandian, Marianna; Mallampalli, Rama K (2013) Surfactant phospholipid metabolism. Biochim Biophys Acta 1831:612-25
Mallampalli, Rama K; Glasser, Jennifer R; Coon, Tiffany A et al. (2013) Calmodulin protects Aurora B on the midbody to regulate the fidelity of cytokinesis. Cell Cycle 12:663-73

Showing the most recent 10 out of 33 publications