Bill B. Chen

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.

A combinatorial F box protein directed pathway controls TRAF adaptor stability to regulate inflammation

Uncontrolled activation of tumor necrosis factor receptor–associated factor (TRAF) proteins may result in profound tissue injury by linking surface signals to cytokine release. Here we show that a ubiquitin E3 ligase component, Fbxo3, potently stimulates cytokine secretion from human inflammatory cells by destabilizing a sentinel TRAF inhibitor, Fbxl2. Fbxo3 and TRAF protein in circulation positively correlated with cytokine responses in subjects with sepsis, and we identified a polymorphism in human Fbxo3, with one variant being hypofunctional. A small-molecule inhibitor targeting Fbxo3 was sufficient to lessen severity of cytokine-driven inflammation in several mouse disease models. These studies identified a pathway of innate immunity that may be useful to detect subjects with altered immune responses during critical illness or provide a basis for therapeutic intervention targeting TRAF protein abundance.

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.

Masking of a Nuclear Signal Motif by Monoubiquitination Leads to Mislocalization and Degradation of the Regulatory Enzyme Cytidylyltransferase

ABSTRACT Monoubiquitination aids in the nuclear export and entrance of proteins into the lysosomal degradative pathway, although the mechanisms are unknown. Cytidylyltransferase (CCTα) is a proteolytically sensitive lipogenic enzyme containing an NH2-terminal nuclear localization signal (NLS). We show here that CCTα is monoubiquitinated at a molecular site (K57) juxtaposed near its NLS, resulting in disruption of its interaction with importin-α, nuclear exclusion, and subsequent degradation within the lysosome. Cellular expression of a CCTα-ubiquitin fusion protein that mimics the monoubiquitinated enzyme resulted in cytoplasmic retention. A CCTα K57R mutant exhibited an extended half-life, was retained in the nucleus, and displayed proteolytic resistance. Importantly, by using CCTα-ubiquitin hybrid constructs that vary in the intermolecular distance between ubiquitin and the NLS, we show that CCTα monoubiquitination masks its NLS, resulting in cytoplasmic retention. These results unravel a unique molecular mechanism whereby monoubiquitination governs the trafficking and life span of a critical regulatory enzyme in vivo.

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.

Calmodulin Antagonizes a Calcium-Activated SCF Ubiquitin E3 Ligase Subunit, FBXL2, To Regulate Surfactant Homeostasis

ABSTRACT Calmodulin is a universal calcium-sensing protein that has pleiotropic effects. Here we show that calmodulin inhibits a new SCF (Skp1–Cullin–F-box) E3 ligase component, FBXL2. During Pseudomonas aeruginosa infection, SCF (FBXL2) targets the key enzyme, CCTα, for its monoubiquitination and degradation, thereby reducing synthesis of the indispensable membrane and surfactant component, phosphatidylcholine. P. aeruginosa triggers calcium influx and calcium-dependent activation of FBXL2 within the Golgi complex, where it engages CCTα. FBXL2 through its C terminus binds to the CCTα IQ motif. FBXL2 knockdown increases CCTα levels and phospholipid synthesis. The molecular interaction of FBXL2 with CCTα is opposed by calmodulin, which traffics to the Golgi complex, binds FBXL2 (residues 80 to 90) via its C terminus, and vies with the ligase for occupancy within the IQ motif. These observations were recapitulated in murine models of P. aeruginosa-induced surfactant deficiency, where calmodulin gene transfer reduced FBXL2 actions by stabilizing CCTα and lessening the severity of inflammatory lung injury. The results provide a unique model of calcium-regulated intermolecular competition between an E3 ligase subunit and an antagonist that is critically relevant to pneumonia and lipid homeostasis.

F-box protein FBXL2 exerts human lung tumor suppressor-like activity by ubiquitin-mediated degradation of cyclin D3 resulting in cell cycle arrest

Dysregulated behavior of cell cycle proteins and their control by ubiquitin E3 ligases is an emerging theme in human lung cancer. Here, we identified and characterized the activity of a novel F-box protein, termed FBXL2, belonging to the SCF (Skip-Cullin1-F-box protein) E3 ligase family. Ectopically expressed FBXL2 triggered G2/M-phase arrest, induced chromosomal anomalies and increased apoptosis of transformed lung epithelia by mediating polyubiquitination and degradation of the mitotic regulator, cyclin D3. Unlike other F-box proteins that target phosphodegrons within substrates, FBXL2 uniquely recognizes a canonical calmodulin (CaM)-binding motif within cyclin D3 to facilitate its polyubiquitination. CaM bound and protected cyclin D3 from FBXL2 by direct intermolecular competition with the F-box protein for access within this motif. The chemotherapeutic agent vinorelbine increased apoptosis of human lung carcinoma cells by inducing FBXL2 expression and cyclin D3 degradation, an effect accentuated by CaM knockdown. Depletion of endogenous FBXL2 stabilized cyclin D3 levels, accelerated cancer cell growth and increased cell viability after vinorelbine treatment. Last, ectopic expression of FBXL2 significantly inhibited the growth and migration of tumorogenic cells and tumor formation in athymic nude mice. These observations implicate SCFFBXL2 as an indispensible regulator of mitosis that serves as a tumor suppressor.

LPS Impairs Phospholipid Synthesis by Triggering β-Transducin Repeat-containing Protein (β-TrCP)-mediated Polyubiquitination and Degradation of the Surfactant Enzyme Acyl-CoA:Lysophosphatidylcholine Acyltransferase I (LPCAT1)

Acyl-CoA:lysophosphatidylcholine acyltransferase 1 (LPCAT1) is a relatively newly described and yet indispensable enzyme needed for generation of the bioactive surfactant phospholipid, dipalmitoylphosphatidylcholine (DPPtdCho). Here, we show that lipopolysaccharide (LPS) causes LPCAT1 degradation using the Skp1-Cullin-F-box ubiquitin E3 ligase component, β-transducin repeat-containing protein (β-TrCP), that polyubiquitinates LPCAT1, thereby targeting the enzyme for proteasomal degradation. LPCAT1 was identified as a phosphoenzyme as Ser178 within a phosphodegron was identified as a putative molecular recognition site for glycogen synthase kinase-3β (GSK-3β) phosphorylation that recruits β-TrCP docking within the enzyme. β-TrCP ubiquitinates LPCAT1 at an acceptor site (Lys221), as substitution of Lys221 with Arg abrogated LPCAT1 polyubiquitination. LPS profoundly reduced immunoreactive LPCAT1 levels and impaired lung surfactant mechanics, effects that were overcome by siRNA to β-TrCP and GSK-3β or LPCAT1 gene transfer, respectively. Thus, LPS appears to destabilize the LPCAT1 protein by GSK-3β-mediated phosphorylation within a canonical phosphodegron for β-TrCP docking and site-specific ubiquitination. LPCAT1 is the first lipogenic substrate for β-TrCP, and the results suggest that modulation of the GSK-3β-SCFβTrCP E3 ligase effector pathway might be a unique strategy to optimize dipalmitoylphosphatidylcholine levels in sepsis.

Lipopolysaccharide Primes the NALP3 Inflammasome by Inhibiting Its Ubiquitination and Degradation Mediated by the SCFFBXL2 E3 Ligase

Background: LPS increases NALP3 levels, but the mechanisms remain unknown. Results: LPS prolongs the lifespan of NALP3 protein by reducing E3 ligase (SCFFBXL2)-mediated ubiquitination. Conclusion: Proinflammatory cytokine release is reduced by a small molecule that restores cellular SCFFBXL2 levels. Significance: We identified a novel pathway of inflammasome priming that may serve as a springboard for future translational studies. The inflammasome is a multiprotein complex that augments the proinflammatory response by increasing the generation and cellular release of key cytokines. Specifically, the NALP3 inflammasome requires two-step signaling, priming and activation, to be functional to release the proinflammatory cytokines IL-1β and IL-18. The priming process, through unknown mechanisms, increases the protein levels of NALP3 and pro-IL-1β in cells. Here we show that LPS increases the NALP3 protein lifespan without significantly altering steady-state mRNA in human cells. LPS exposure reduces the ubiquitin-mediated proteasomal processing of NALP3 by inducing levels of an E3 ligase component, FBXO3, which targets FBXL2. The latter is an endogenous mediator of NALP3 degradation. FBXL2 recognizes Trp-73 within NALP3 for interaction and targets Lys-689 within NALP3 for ubiquitin ligation and degradation. A unique small molecule inhibitor of FBXO3 restores FBXL2 levels, resulting in decreased NALP3 protein levels in cells and, thereby, reducing the release of IL-1β and IL-18 in human inflammatory cells after NALP3 activation. Our findings uncover NALP3 as a molecular target for FBXL2 and suggest that therapeutic targeting of the inflammasome may serve as a platform for preclinical intervention.