Tiffany A. Coon

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.

Let-7d microRNA affects mesenchymal phenotypic properties of lung fibroblasts

MicroRNAs are small noncoding RNAs that inhibit protein expression. We have previously shown that the inhibition of the microRNA let-7d in epithelial cells caused changes consistent with epithelial-to-mesenchymal transition (EMT) both in vitro and in vivo. The aim of this study was to determine whether the introduction of let-7d into fibroblasts alters their mesenchymal properties. Transfection of primary fibroblasts with let-7d caused a decrease in expression of the mesenchymal markers α-smooth muscle actin, N-cadherin, fibroblast-specific protein-1, and fibronectin, as well as an increase in the epithelial markers tight junction protein-1 and keratin 19. Phenotypic changes were also present, including a delay in wound healing, reduced motility, and proliferation of fibroblasts following transfection. In addition, we examined the effects of transfection on fibroblast responsiveness to TGF-β, an important factor in many fibrotic processes such as lung fibrosis and found that let-7d transfection significantly attenuated high-mobility group-A2 protein induction by TGF-β. Our results indicate that administration of the epithelial microRNA let-7d can significantly alter the phenotype of primary fibroblasts.

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.

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.

SCF E3 ligase F-box protein complex SCFFBXL19 regulates cell migration by mediating Rac1 ubiquitination and degradation

Rac1, a member of the Rho family of GTPases, regulates diverse cellular functions, including cytoskeleton reorganization and cell migration. F‐box proteins are major subunits within the Skp1‐Cul1‐F‐box (SCF) E3 ubiquitin ligases that recognize specific substrates for ubiquitination. The role of F‐box proteins in regulating Rac1 stability has not been studied. Mouse lung epithelial (MLE12) cells were used to investigate Rac1 stability and cell migration. Screening of an F‐box protein library and in vitro ubiquitination assays identified FBXL19, a relatively new member of the F‐box protein family that targets Rac1 for its polyubiquitination and proteasomal degradation. Overexpression of FBXL19 decreased both Rac1 active and inactive forms and significantly reduced cellular migration. Protein kinase AKT‐mediated phosphorylation of Rac1 at serine71 was essential for FBXL19‐mediated Rac1 ubiquitination and depletion. Lysin166 within Rac1 was identified as a polyubiquitination acceptor site. Rac1S71A and Rac1K166R mutant proteins were resistant to FBXL19‐mediated ubiquitination and degradation. Further, ectopically expressed FBXL19 reduced cell migration in Rac1‐overexpressing cells (P<0.01, Rac1 cells vs. FBXL19+Rac1 cells), but not in Rac1 lysine166 mutantoverexpressing cells. FBXL19 diminished formation of the migratory leading edge. Thus, SCFFBXL19 targets Rac1 for its disposal, a process regulated by AKT. These findings provide the first evidence of an F‐box protein targeting a small G protein for ubiquitination and degradation to modulate cell migration.—Zhao, J., Mialki, R. K., Wei, J., Coon, T. A., Zou, C., Chen, B. B., Mallampalli, R. K., Zhao, Y. SCF E3 ligase F‐box protein complex SCF regulates cell migration by mediating Rac1 ubiquitination and degradation. FASEB J. 27, 2611‐2619 (2013). www.fasebj.org

FBXL2 is a ubiquitin E3 ligase subunit that triggers mitotic arrest.

Mitotic progression is regulated by ubiquitin E3 ligase complexes to carefully orchestrate eukaryotic cell division. Here, we show that a relatively new E3 ligase component belonging to the SCF (Skip-Cullin1-F-box protein) E3 ligase family, SCFFBXL2, impairs cell proliferation by mediating cyclin D3 polyubiquitination and degradation. Both cyclin D3 and FBXL2 colocalize within the centrosome. FBXL2 overexpression led to G2/M-phase arrest in transformed epithelia, resulting in the appearance of supernumerary centrosomes, tetraploidy and nuclei where condensed chromosomes are arranged on circular monopolar spindles typical of mitotic arrest. RNAi-mediated knockdown of cyclin D3 recapitulated effects of SCFFBXL2 expression. SCFFBXL2 impaired the ability of cyclin D3 to associate with centrosomal assembly proteins [Aurora A, polo-like kinase 4 (Plk4), CDK11]. Thus, these results suggest a role for SCFFBXL2 in regulating the fidelity of cellular division.

Targeting F Box Protein Fbxo3 To Control Cytokine-Driven Inflammation

Cytokine-driven inflammation underlies the pathobiology of a wide array of infectious and immune-related disorders. The TNFR-associated factor (TRAF) proteins have a vital role in innate immunity by conveying signals from cell surface receptors to elicit transcriptional activation of genes encoding proinflammatory cytokines. We discovered that a ubiquitin E3 ligase F box component, termed Fbxo3, potently stimulates cytokine secretion from human inflammatory cells by mediating the degradation of the TRAF inhibitory protein, Fbxl2. Analysis of the Fbxo3 C-terminal structure revealed that the bacterial-like ApaG molecular signature was indispensible for mediating Fbxl2 disposal and stimulating cytokine secretion. By targeting this ApaG motif, we developed a highly unique, selective genus of small-molecule Fbxo3 inhibitors that by reducing TRAF protein levels, potently inhibited cytokine release from human blood mononuclear cells. The Fbxo3 inhibitors effectively lessened the severity of viral pneumonia, septic shock, colitis, and cytokine-driven inflammation systemically in murine models. Thus, pharmacological targeting of Fbxo3 might be a promising strategy for immune-related disorders characterized by a heightened host inflammatory response.