Rama K. Mallampalli

PLUNC Is a Novel Airway Surfactant Protein with Anti-Biofilm Activity

Background The PLUNC (“Palate, lung, nasal epithelium clone”) protein is an abundant secretory product of epithelia present throughout the conducting airways of humans and other mammals, which is evolutionarily related to the lipid transfer/lipopolysaccharide binding protein (LT/LBP) family. Two members of this family - the bactericidal/permeability increasing protein (BPI) and the lipopolysaccharide binding protein (LBP) - are innate immune molecules with recognized roles in sensing and responding to Gram negative bacteria, leading many to propose that PLUNC may play a host defense role in the human airways. Methodology/Principal Findings Based on its marked hydrophobicity, we hypothesized that PLUNC may be an airway surfactant. We found that purified recombinant human PLUNC greatly enhanced the ability of aqueous solutions to spread on a hydrophobic surface. Furthermore, we discovered that PLUNC significantly reduced surface tension at the air-liquid interface in aqueous solutions, indicating novel and biologically relevant surfactant properties. Of note, surface tensions achieved by adding PLUNC to solutions are very similar to measurements of the surface tension in tracheobronchial secretions from humans and animal models. Because surfactants of microbial origin can disperse matrix-encased bacterial clusters known as biofilms [1], we hypothesized that PLUNC may also have anti-biofilm activity. We found that, at a physiologically relevant concentration, PLUNC inhibited biofilm formation by the airway pathogen Pseudomonas aeruginosa in an in vitro model. Conclusions/Significance Our data suggest that the PLUNC protein contributes to the surfactant properties of airway secretions, and that this activity may interfere with biofilm formation by an airway pathogen.

Dynamic regulation of cardiolipin by the lipid pump Atp8b1 determines the severity of lung injury in experimental pneumonia

Pneumonia remains the leading cause of death from infection in the US, yet fundamentally new conceptual models underlying its pathogenesis have not emerged. We show that humans and mice with bacterial pneumonia have markedly elevated amounts of cardiolipin, a rare, mitochondrial-specific phospholipid, in lung fluid and find that it potently disrupts surfactant function. Intratracheal cardiolipin administration in mice recapitulates the clinical phenotype of pneumonia, including impaired lung mechanics, modulation of cell survival and cytokine networks and lung consolidation. We have identified and characterized the activity of a unique cardiolipin transporter, the P-type ATPase transmembrane lipid pump Atp8b1, a mutant version of which is associated with severe pneumonia in humans and mice. Atp8b1 bound and internalized cardiolipin from extracellular fluid via a basic residue–enriched motif. Administration of a peptide encompassing the cardiolipin binding motif or Atp8b1 gene transfer in mice lessened bacteria-induced lung injury and improved survival. The results unveil a new paradigm whereby Atp8b1 is a cardiolipin importer whose capacity to remove cardiolipin from lung fluid is exceeded during inflammation or when Atp8b1 is defective. This discovery opens the door for new therapeutic strategies directed at modulating the abundance or molecular interactions of cardiolipin in pneumonia.

The Acute Respiratory Distress Syndrome: From Mechanism to Translation

The acute respiratory distress syndrome (ARDS) is a form of severe hypoxemic respiratory failure that is characterized by inflammatory injury to the alveolar capillary barrier, with extravasation of protein-rich edema fluid into the airspace. Although many modalities to treat ARDS have been investigated over the past several decades, supportive therapies remain the mainstay of treatment. In this article, we briefly review the definition, epidemiology, and pathophysiology of ARDS and present emerging aspects of ARDS pathophysiology that encompass modulators of the innate immune response, damage signals, and aberrant proteolysis that may serve as a foundation for future therapeutic targets.

Surfactant phospholipid metabolism.

Pulmonary surfactant is essential for life and is composed of a complex lipoprotein-like mixture that lines the inner surface of the lung to prevent alveolar collapse at the end of expiration. The molecular composition of surfactant depends on highly integrated and regulated processes involving its biosynthesis, remodeling, degradation, and intracellular trafficking. Despite its multicomponent composition, the study of surfactant phospholipid metabolism has focused on two predominant components, disaturated phosphatidylcholine that confers surface-tension lowering activities, and phosphatidylglycerol, recently implicated in innate immune defense. Future studies providing a better understanding of the molecular control and physiological relevance of minor surfactant lipid components are needed. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.

Lung macrophages "digest" carbon nanotubes using a superoxide/peroxynitrite oxidative pathway.

In contrast to short-lived neutrophils, macrophages display persistent presence in the lung of animals after pulmonary exposure to carbon nanotubes. While effective in the clearance of bacterial pathogens and injured host cells, the ability of macrophages to “digest” carbonaceous nanoparticles has not been documented. Here, we used chemical, biochemical, and cell and animal models and demonstrated oxidative biodegradation of oxidatively functionalized single-walled carbon nanotubes via superoxide/NO* → peroxynitrite-driven oxidative pathways of activated macrophages facilitating clearance of nanoparticles from the lung.

Emerging therapies targeting the ubiquitin proteasome system in cancer

The ubiquitin proteasome system (UPS) is an essential metabolic constituent of cellular physiology that tightly regulates cellular protein concentrations with specificity and precision to optimize cellular function. Inhibition of the proteasome has proven very effective in the treatment of multiple myeloma, and this approach is being tested for utility in other malignancies. New pharmaceuticals targeting the proteasome itself or specific proximal pathways of the UPS are in development as antiproliferatives or immunomodulatory agents. In this article, we discuss the biology of UPS-targeting drugs, their use as therapy for neoplasia, and the state of clinical and preclinical development for emerging therapeutics.

14-3-3 Binding and Phosphorylation of Neuroglobin during Hypoxia Modulate Six-to-Five Heme Pocket Coordination and Rate of Nitrite Reduction to Nitric Oxide

Background: Neuroglobin protects neurons from hypoxia; however, the underlying mechanisms for this effect remain poorly understood. Results: Hypoxia increases neuroglobin phosphorylation, binding to 14-3-3, and nitrite reduction to form nitric oxide. Conclusion: Hypoxia-dependent post-translational modifications to neuroglobin regulate the six-to-five heme pocket equilibrium and heme access to ligands. Significance: Hypoxia-regulated neuroglobin may contribute to the cellular adaptation to hypoxia. Neuroglobin protects neurons from hypoxia in vitro and in vivo; however, the underlying mechanisms for this effect remain poorly understood. Most of the neuroglobin is present in a hexacoordinate state with proximal and distal histidines in the heme pocket directly bound to the heme iron. At equilibrium, the concentration of the five-coordinate neuroglobin remains very low (0.1–5%). Recent studies have shown that post-translational redox regulation of neuroglobin surface thiol disulfide formation increases the open probability of the heme pocket and allows nitrite binding and reaction to form NO. We hypothesized that the equilibrium between the six- and five-coordinate states and secondary reactions with nitrite to form NO could be regulated by other hypoxia-dependent post-translational modification(s). Protein sequence models identified candidate sites for both 14-3-3 binding and phosphorylation. In both in vitro experiments and human SH-SY5Y neuronal cells exposed to hypoxia and glucose deprivation, we observed that 1) neuroglobin phosphorylation and protein-protein interactions with 14-3-3 increase during hypoxic and metabolic stress; 2) neuroglobin binding to 14-3-3 stabilizes and increases the half-life of phosphorylation; and 3) phosphorylation increases the open probability of the heme pocket, which increases ligand binding (CO and nitrite) and accelerates the rate of anaerobic nitrite reduction to form NO. These data reveal a series of hypoxia-dependent post-translational modifications to neuroglobin that regulate the six-to-five heme pocket equilibrium and heme access to ligands. Hypoxia-regulated reactions of nitrite and neuroglobin may contribute to the cellular adaptation to hypoxia.

F box protein FBXL2 targets cyclin D2 for ubiquitination and degradation to inhibit leukemic cell proliferation

Hematologic maligancies exhibit a growth advantage by up-regulation of components within the molecular apparatus involved in cell-cycle progression. The SCF (Skip-Cullin1-F-box protein) E3 ligase family provides homeostatic feedback control of cell division by mediating ubiquitination and degradation of cell-cycle proteins. By screening several previously undescribed E3 ligase components, we describe the behavior of a relatively new SCF subunit, termed FBXL2, that ubiquitinates and destabilizes cyclin D2 protein leading to G(0) phase arrest and apoptosis in leukemic and B-lymphoblastoid cell lines. FBXL2 expression was strongly suppressed, and yet cyclin D2 protein levels were robustly expressed in acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) patient samples. Depletion of endogenous FBXL2 stabilized cyclin D2 levels, whereas ectopically expressed FBXL2 decreased cyclin D2 lifespan. FBXL2 did not bind a phosphodegron within its substrate, which is typical of other F-box proteins, but uniquely targeted a calmodulin-binding signature within cyclin D2 to facilitate its polyubiquitination. Calmodulin competes with the F-box protein for access to this motif where it bound and protected cyclin D2 from FBXL2. Calmodulin reversed FBXL2-induced G(0) phase arrest and attenuated FBXL2-induced apoptosis of lymphoblastoid cells. These results suggest an antiproliferative effect of SCF(FBXL2) in lymphoproliferative malignancies.

Acyl-CoA:Lysophosphatidylcholine Acyltransferase I (Lpcat1) Catalyzes Histone Protein O-Palmitoylation to Regulate mRNA Synthesis

The enzyme acyl-CoA:lysophosphatidylcholine acyltransferase (Lpcat1) is a critical cytosolic enzyme needed for lung surfactant synthesis that catalyzes an acyltransferase reaction by adding a palmitate to the sn-2 position of lysophospholipids. Here we report that histone H4 protein is subject to palmitoylation catalyzed by Lpcat1 in a calcium-regulated manner. Cytosolic Lpcat1 was observed to shift into the nucleus in lung epithelia in response to exogenous Ca2+. Nuclear Lpcat1 colocalizes with and binds to histone H4, where it catalyzes histone H4 palmitoylation. Mutagenesis studies demonstrated that Ser47 within histone H4 serves as a putative acceptor site, indicative of Lpcat1-mediated O-palmitoylation. Lpcat1 knockdown or expression of a histone H4 Ser47A mutant protein in cells decreased cellular mRNA synthesis. These findings provide the first evidence of a protein substrate for Lpcat1 and reveal that histone lipidation may occur through its O-palmitoylation as a novel post-translational modification. This epigenetic modification regulates global gene transcriptional activity.

SPLUNC1/BPIFA1 Contributes to Pulmonary Host Defense against Klebsiella pneumoniae Respiratory Infection

Epithelial host defense proteins comprise a critical component of the pulmonary innate immune response to infection. The short palate, lung, nasal epithelium clone (PLUNC) 1 (SPLUNC1) protein is a member of the bactericidal/permeability-increasing (BPI) fold-containing (BPIF) protein family, sharing structural similarities with BPI-like proteins. SPLUNC1 is a 25 kDa secretory protein that is expressed in nasal, oropharyngeal, and lung epithelia, and has been implicated in airway host defense against Pseudomonas aeruginosa and other organisms. SPLUNC1 is reported to have surfactant properties, which may contribute to anti-biofilm defenses. The objective of this study was to assess the importance of SPLUNC1 surfactant activity in airway epithelial secretions and to explore its biological relevance in the context of a bacterial infection model. Using cultured airway epithelia, we confirmed that SPLUNC1 is critically important for maintenance of low surface tension in airway fluids. Furthermore, we demonstrated that recombinant SPLUNC1 (rSPLUNC1) significantly inhibited Klebsiella pneumoniae biofilm formation on airway epithelia. We subsequently found that Splunc1(-/-) mice were significantly more susceptible to infection with K. pneumoniae, confirming the likely in vivo relevance of this anti-biofilm effect. Our data indicate that SPLUNC1 is a crucial component of mucosal innate immune defense against pulmonary infection by a relevant airway pathogen, and provide further support for the novel hypothesis that SPLUNC1 protein prevents bacterial biofilm formation through its ability to modulate surface tension of airway fluids.