Travis Lear

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.

E3 Ligase Subunit Fbxo15 and PINK1 Kinase Regulate Cardiolipin Synthase 1 Stability and Mitochondrial Function in Pneumonia

SUMMARY Acute lung injury (ALI) is linked to mitochondrial injury, resulting in impaired cellular oxygen utilization; however, it is unknown how these events are linked on the molecular level. Cardiolipin, a mitochondrial-specific lipid, is generated by cardiolipin synthase (CLS1). Here, we show that S. aureus activates a ubiquitin E3 ligase component, Fbxo15, that is sufficient to mediate proteasomal degradation of CLS1 in epithelia, resulting in decreased cardiolipin availability and disrupted mitochondrial function. CLS1 is destabilized by the phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), which binds CLS1 to phosphorylate and regulates CLS1 disposal. Like Fbxo15, PINK1 interacts with and regulates levels of CLS1 through a mechanism dependent upon Thr219. S. aureus infection upregulates this Fbxo15-PINK1 pathway to impair mitochondrial integrity, and Pink1 knockout mice are less prone to S. aureus-induced ALI. Thus, ALI-associated disruption of cellular bioenergetics involves bioeffectors that utilize a phosphodegron to elicit ubiquitin-mediated disposal of a key mitochondrial enzyme.

F-box protein Fbxl18 mediates polyubiquitylation and proteasomal degradation of the pro-apoptotic SCF subunit Fbxl7

Fbxl7, a subunit of the SCF (Skp-Cul1-F-box protein) complex induces mitotic arrest in cells; however, molecular factors that control its cellular abundance remain largely unknown. Here, we identified that an orphan F-box protein, Fbxl18, targets Fbxl7 for its polyubiquitylation and proteasomal degradation. Lys 109 within Fbxl7 is an essential acceptor site for ubiquitin conjugation by Fbxl18. An FQ motif within Fbxl7 serves as a molecular recognition site for Fbxl18 interaction. Ectopically expressed Fbxl7 induces apoptosis in Hela cells, an effect profoundly accentuated after cellular depletion of Fbxl18 protein or expression of Fbxl7 plasmids encoding mutations at either Lys 109 or within the FQ motif. Ectopic expression of Fbxl18 plasmid-limited apoptosis caused by overexpressed Fbxl7 plasmid. Thus, Fbxl18 regulates apoptosis by mediating ubiquitin-dependent proteasomal degradation of the pro-apoptotic protein Fbxl7 that may impact cellular processes involved in cell cycle progression.

The proinflammatory role of HECTD2 in innate immunity and experimental lung injury.

Antagonizing the E3 ligase HECTD2 reduces inflammation severity and experimental lung injury. Calming the cytokine storm The innate immune response is poised to act quickly in the face of pathogenic invaders; however, this priming may lead to a cytokine storm, where excessive production of proinflammatory cytokines harms the host. Coon et al. now report that HECTD2, a ubiquitin E3 ligase, can degrade the anti-inflammatory protein PIAS1, increasing this inflammatory effect. Indeed, a small-molecule inhibitor of HECTD2 reduced lung inflammation in a mouse model. Moreover, people with a polymorphism in HECTD2 had lower inflammation and were protected from acute respiratory distress syndrome. Thus, HECTD2 serves as a new therapeutic target for inflammation-induced lung injury. Invading pathogens may trigger overactivation of the innate immune system, which results in the release of large amounts of proinflammatory cytokines (cytokine storm) and leads to the development of pulmonary edema, multiorgan failure, and shock. PIAS1 is a multifunctional and potent anti-inflammatory protein that negatively regulates several key inflammatory pathways such as Janus kinase (JAK)–signal transducer and activator of transcription (STAT) and nuclear factor κB (NF-κB). We discovered a ubiquitin E3 ligase, HECTD2, which ubiquitinated and mediated the degradation of PIAS1, thus increasing inflammation in an experimental pneumonia model. We found that GSK3β phosphorylation of PIAS1 provided a phosphodegron for HECTD2 targeting. We also identified a mislocalized HECTD2 polymorphism, HECTD2A19P, that was present in 8.5% of the population and functioned to reduce inflammation. This polymorphism prevented HECTD2/PIAS1 nuclear interaction, thus preventing PIAS1 degradation. The HECTD2A19P polymorphism was also protective toward acute respiratory distress syndrome (ARDS). We then developed a small-molecule inhibitor, BC-1382, that targeted HECTD2 and attenuated lipopolysaccharide (LPS)– and Pseudomonas aeruginosa–induced lung inflammation. These studies describe an unreported innate immune pathway and suggest that mutation or antagonism of the E3 ligase HECTD2 results in reduced severity of lung inflammation by selectively modulating the abundance of the anti-inflammatory protein PIAS1.

The Proapoptotic F-box Protein Fbxl7 Regulates Mitochondrial Function by Mediating the Ubiquitylation and Proteasomal Degradation of Survivin

Background: The SCF ubiquitin E3 ligase component Fbxl7 possesses proapoptotic activity. Results: Fbxl7 targets the antiapoptotic protein survivin for polyubiquitylation and proteasomal degradation. Conclusion: Survivin protects mitochondria from damage induced by Fbxl7. Significance: Understanding how F-box proteins regulate survivin might impact therapies to preserve cellular bioenergetics. Fbxl7, a component of the Skp1·Cul1·F-box protein type ubiquitin E3 ligase, regulates mitotic cell cycle progression. Here we demonstrate that overexpression of Fbxl7 in lung epithelia decreases the protein abundance of survivin, a member of the inhibitor of apoptosis family. Fbxl7 mediates polyubiquitylation and proteasomal degradation of survivin by interacting with Glu-126 within its carboxyl-terminal α helix. Furthermore, both Lys-90 and Lys-91 within survivin serve as ubiquitin acceptor sites. Ectopically expressed Fbxl7 impairs mitochondrial function, whereas depletion of Fbxl7 protects mitochondria from actions of carbonyl cyanide m-chlorophenylhydrazone, an inhibitor of oxidative phosphorylation. Compared with wild-type survivin, cellular expression of a survivin mutant protein deficient in its ability to interact with Fbxl7 (E126A) and a ubiquitylation-resistant double point mutant (KK90RR/KK91RR) rescued mitochondria to a larger extent from damage induced by overexpression of Fbxl7. Therefore, these data suggest that the Skp1·Cul1·F-box protein complex subunit Fbxl7 modulates mitochondrial function by controlling the cellular abundance of survivin. The results raise opportunities for F-box protein targeting to preserve mitochondrial function.

Novel PDE4 inhibitors derived from Chinese medicine forsythia.

Cyclic adenosine monophosphate (cAMP) is a crucial intracellular second messenger molecule that converts extracellular molecules to intracellular signal transduction pathways generating cell- and stimulus-specific effects. Importantly, specific phosphodiesterase (PDE) subtypes control the amplitude and duration of cAMP-induced physiological processes and are therefore a prominent pharmacological target currently used in a variety of fields. Here we tested the extracts from traditional Chinese medicine, Forsythia suspense seeds, which have been used for more than 2000 years to relieve respiratory symptoms. Using structural-functional analysis we found its major lignin, Forsynthin, acted as an immunosuppressant by inhibiting PDE4 in inflammatory and immune cell. Moreover, several novel, selective small molecule derivatives of Forsythin were tested in vitro and in murine models of viral and bacterial pneumonia, sepsis and cytokine-driven systemic inflammation. Thus, pharmacological targeting of PDE4 may be a promising strategy for immune-related disorders characterized by amplified host inflammatory response.

RING finger E3 ligase PPP1R11 regulates TLR2 signaling and innate immunity

Toll-like receptor 2 (TLR2) is a pattern recognition receptor that recognizes many types of PAMPs that originate from gram-positive bacteria. Here we describe a novel mechanism regulating TLR2 protein expression and subsequent cytokine release through the ubiquitination and degradation of the receptor in response to ligand stimulation. We show a new mechanism in which an uncharacterized RING finger E3 ligase, PPP1R11, directly ubiquitinates TLR2 both in vitro and in vivo, which leads to TLR2 degradation and disruption of the signaling cascade. Lentiviral gene transfer or knockdown of PPP1R11 in mouse lungs significantly affects lung inflammation and the clearance of Staphylococcus aureus. There is a negative correlation between PPP1R11 and TLR2 levels in white blood cell samples isolated from patients with Staphylococcus aureus infections. These results suggest that PPP1R11 plays an important role in regulating innate immunity and gram-positive bacterial clearance by functioning, in part, through the ubiquitination and degradation of TLR2. DOI: http://dx.doi.org/10.7554/eLife.18496.001

Therapeutic targets in fibrotic pathways.

The pathogenetic heterogeneity of pulmonary fibrosis yields both challenges and opportunities for therapy. Its complexity implicates a variety of cellular processes, signaling pathways, and genetics as drivers of disease. TGF-β stimulation is one avenue, and is central to pro-fibrotic protein expression, leading to decreased pulmonary function. Here we report our recent findings, introducing the E3 ligase Fibrosis Inducing E3 Ligase 1 (FIEL1) as an important regulator of TGF-β signaling through the selective degradation of PIAS4. FIEL1 exacerbates bleomycin-induced murine pulmonary fibrosis, while its silencing attenuates the fibrotic phenotype. Further, we developed a small molecule inhibitor of FIEL1 (BC-1485) that inhibits the degradation of PIAS4, and ameliorates fibrosis in murine models. New understanding of this pathway illustrates the many targeting opportunities among the complexity of pulmonary fibrosis in the continuing search for therapy.

The Human IL-22 Receptor Is Regulated through the Action of the Novel E3 Ligase Subunit FBXW12, Which Functions as an Epithelial Growth Suppressor

Interleukin- (IL-) 22 signaling is protective in animal models of pneumonia and bacteremia by Klebsiella pneumoniae and mediates tissue recovery from influenza and Staph aureus infection. We recently described processing of mouse lung epithelial IL-22 receptor (IL-22R) by ubiquitination on the intracellular C-terminal. To identify cellular factors that regulate human IL-22R, we screened receptor abundance while overexpressing constituents of the ubiquitin system and identify that IL-22R can be shuttled for degradation by multiple previously uncharacterized F-box protein E3 ligase subunits. We observe that in human cells IL-22R is destabilized by FBXW12. FBXW12 causes depletion of endogenous and plasmid-derived IL-22R in lung epithelia, binds the E3 ligase constituent Skp-1, and facilitates ubiquitination of IL-22R in vitro. FBXW12 knockdown with shRNA increases IL-22R abundance and STAT3 phosphorylation in response to IL-22 cytokine treatment. FBXW12 shRNA increases human epithelial cell growth and cell cycle progression with enhanced constitutive activity of map kinases JNK and ERK. These findings indicate that the heretofore-undescribed protein FBXW12 functions as an E3 ligase constituent to ubiquitinate and degrade IL-22R and that therapeutic FBXW12 inhibition may enhance IL-22 signaling and bolster mucosal host defense and infection containment.

Receptor for advanced glycation end products is targeted by FBXO10 for ubiquitination and degradation

The receptor for advanced glycation end products (RAGE) is a highly expressed cell membrane receptor serving to anchor lung epithelia to matrix components, and it also amplifies inflammatory signaling during acute lung injury. However, mechanisms that regulate its protein concentrations in cells remain largely unknown. Here we show that RAGE exhibits an extended life span in lung epithelia (t½ 6 h), is monoubiquitinated at K374, and is degraded in lysosomes. The RAGE ligand ODN2006, a synthetic oligodeoxynucleotide resembling pathogenic hypomethylated CpG DNA, promotes rapid lysosomal RAGE degradation through activation of protein kinase Cζ (PKCζ), which phosphorylates RAGE. PKCζ overexpression enhances RAGE degradation, while PKCζ knockdown stabilizes RAGE protein levels and prevents ODN2006‐mediated degradation. We identify that RAGE is targeted by the ubiquitin E3 ligase subunit F‐box protein O10 (FBXO10), which associates with RAGE to mediate its ubiquitination and degradation. FBXO10 depletion in cells stabilizes RAGE and is required for ODN2006‐mediated degradation. These data suggest that modulation of regulators involved in ubiquitin‐mediated disposal of RAGE might serve as unique molecular inputs directing RAGE cellular concentrations and downstream responses, which are critical in an array of inflammatory disorders, including acute lung injury.—Evankovich, J., Lear, T., Mckelvey, A., Dunn, S., Londino, J., Liu, Y., Chen, B. B., Mallampalli, R. K. Receptor for advanced glycation end products is targeted by FBXO10 for ubiquitination and degradation. FASEB J. 31, 3894–3903 (2017). www.fasebj.org—Evankovich, John, Lear, Travis, Mckelvey, Alison, Dunn, Sarah, Londino, James, Liu, Yuan, Chen, Bill B., Mallampalli, Rama K. Receptor for advanced glycation end products is targeted by FBXO10 for ubiquitination and degradation. FASEB J. 31, 3894–3903 (2017)