F-box protein FBXL19-mediated ubiquitination and degradation of the receptor for IL-33 limits pulmonary inflammation.

The ST2L receptor for interleukin 33 (IL-33) mediates pulmonary inflammation and immune system-related disorders, such as asthma and rheumatoid arthritis. At present, very little is known about the molecular regulation of ST2L expression. Here we found that FBXL19, an 'orphan' member of the Skp1-Cullin-F-box family of E3 ubiquitin ligases, selectively bound to ST2L to mediate its polyubiquitination and elimination in the proteasome. Degradation of ST2L involved phosphorylation of ST2L at Ser442 catalyzed by the kinase GSK3ß. Overexpression of FBXL19 abrogated the proapoptotic and inflammatory effects of IL-33 and lessened the severity of lung injury in mouse models of pneumonia. Our results suggest that modulation of the IL-33-ST2L axis by ubiquitin ligases might serve as a unique strategy for lessening pulmonary inflammation.

Cross-Regulation of F-Box Protein FBXL2 with T-bet and TNF-α during Acute and Chronic Lung Allograft Rejection.

Chronic lung allograft dysfunction is the major barrier to long-term survival in lung transplant recipients. Evidence supports type 1 alloimmunity as the predominant response in acute/chronic lung rejection, but the immunoregulatory mechanisms remain incompletely understood. We studied the combinatorial F-box E3 ligase system: F-box protein 3 (FBXO3; proinflammatory) and F-box and leucine-rich repeat protein 2 (FBXL2; anti-inflammatory and regulates TNFR-associated factor [TRAF] protein). Using the mouse orthotopic lung transplant model, we evaluated allografts from BALB/c → C57BL/6 (acute rejection; day 10) and found significant induction of FBXO3 and diminished FBXL2 protein along with elevated T-bet, IFN-γ, and TRAF proteins 1-5 compared with isografts. In the acute model, treatment with costimulation blockade (MR1/CTLA4-Ig) resulted in attenuated FBXO3, preserved FBXL2, and substantially reduced T-bet, IFN-γ, and TRAFs 1-5, consistent with a key role for type 1 alloimmunity. Immunohistochemistry revealed significant changes in the FBXO3/FBXL2 balance in airway epithelia and infiltrating mononuclear cells during rejection compared with isografts or costimulation blockade-treated allografts. In the chronic lung rejection model, DBA/2J/C57BL/6F1 > DBA/2J (day 28), we observed persistently elevated FBXO3/FBXL2 balance and T-bet/IFN-γ protein and similar findings from lung transplant recipient lungs with chronic lung allograft dysfunction versus controls. We hypothesized that FBXL2 regulated T-bet and found FBXL2 was sufficient to polyubiquitinate T-bet and coimmunoprecipitated with T-bet on pulldown experiments and vice versa in Jurkat cells. Transfection with FBXL2 diminished T-bet protein in a dose-dependent manner in mouse lung epithelial cells. In testing type 1 cytokines, TNF-α was found to negatively regulate FBXL2 protein and mRNA levels. Together, our findings show the combinatorial E3 ligase FBXO3/FBXL2 system plays a role in the regulation of T-bet through FBXL2, with negative cross-regulation of TNF-α on FBXL2 during lung allograft rejection.

IL-37-induced activation of glycogen synthase kinase 3ß promotes IL-1R8/Sigirr phosphorylation, internalization, and degradation in lung epithelial cells.

Interleukin (IL)-37 diminishes a variety of inflammatory responses through ligation to its receptor IL-1R8/Sigirr. Sigirr is a Toll like receptor/IL-1R family member. We have shown that Sigirr is not stable in response to IL-37 treatment. IL-37-induced Sigirr degradation is mediated by the ubiquitin-proteasome system, and the process is reversed by a deubiquitinase, USP13. However, the molecular mechanisms by which USP13 regulates Sigirr stability have not been revealed. In this study, we investigate the roles of glycogen synthesis kinase 3ß (GSK3ß) in Sigirr phosphorylation and stability. IL-37 stimulation induced Sigirr phosphorylation and degradation, as well as activation of GSK3ß. Inhibition of GSK3ß attenuated IL-37-induced Sigirr phosphorylation, while exogenous expressed GSK3ß promoted Sigirr phosphorylation at threonine (T)372 residue. Sigirr association with GSK3ß was detected. Amino acid residues 51-101 in GSK3ß were identified as the Sigirr binding domain. These data indicate that GSK3ß mediates IL-37-induced threonine phosphorylation of Sigirr. Further, we investigated the role of GSK3ß-mediated phosphorylation of Sigirr in Sigirr degradation. Inhibition of GSK3ß attenuated IL-37-induced Sigirr degradation, while T372 mutant of Sigirr was resistant to IL-37-mediated degradation. Furthermore, inhibition of Sigirr phosphorylation prevented Sigirr internalization and association with USP13, suggesting GSK3ß promotes Sigirr degradation through disrupting Sigirr association with USP13.

A blocking peptide stabilizes lysophosphatidic acid receptor 1 and promotes lysophosphatidic acid-induced cellular responses.

G protein-coupled receptors regulate a variety of cellular responses and have been considered as therapeutic targets for human diseases. Lysophosphatidic acid receptor 1 (LPA1) is a receptor for bioactive lysophospholipid, LPA. LPA/LPA1-mediated signaling contributes to inflammatory and fibrotic responses in lung diseases; thus understanding regulation of LPA1 stability is important for modulating LPA/LPA1 signaling. Our previous study has shown that LPA1 is degraded in the Nedd4 like (Nedd4L) E3 ubiquitin ligase-mediated ubiquitin-proteasome system. In the current study, we attempt to identify a peptide that stabilizes LPA1 through disrupting LPA1 association with Nedd4L. LPA treatment induces both endogenous and overexpressed LPA1 degradation, which is attenuated by a proteasome inhibitor, suggesting that LPA1 is degraded in the proteasome. LPA increases phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2) and I-κB kinase in lung epithelial cells, and this effect is promoted by overexpression of a peptide (P1) that mimics C-terminal of LPA1. P1, not a control peptide, attenuates LPA-induced LPA1 ubiquitination and degradation, suggesting that P1 stabilizes LPA1. Further, P1 diminishes Nedd4L-mediated degradation of LPA1 and Nedd4L/LPA1 association. In addition to increasing LPA1 signaling, P1 enhances LPA-induced cell migration and gene expression of Elafin, matrix metallopeptidase 1, and serpin family B member 2 in lung epithelial cells. These data suggest that disruption of LPA1 interaction with Nedd4L by P1 increases LPA1 stability and LPA/LPA1 signaling.

SCF FBXW17 E3 ubiquitin ligase regulates FBXL19 stability and cell migration.

The Skp1-Cul1-F-box protein (SCF) E3 ligase complex is one of the largest ubiquitin E3 ligase families. FBXL19, a F-box protein in SCFFBXL19 E3 ligase complex, regulates a variety of cellular responses including cell migration. We have shown that FBXL19 is not stable and its degradation is mediated by the ubiquitin-proteasome system, while the ubiquitin E3 ligase for FBXL19 ubiquitination and degradation has not been identified. In the study, we discovered that a new ubiquitin E3 ligase, SCFFBXW17 , ubiquitinates and induces FBXL19 degradation. Exogenous FBXW17 targets FBXL19 for its ubiquitination and degradation. Lysine 114 in FBXL19 is a potential ubiquitin acceptor site. Acetylation of FBXL19 attenuated SCFFBXW17 -mediated FBXL19 degradation. SCFFBXL19 E3 ligase reduced Rac1 levels and cell migration, while the effects were attenuated by exogenous FBXW17. Downregulation of FBXW17 attenuated lysophosphatidic acid-induced lamellipodia formation and Rac1 accumulation at migration leading edge. Taken together with our previous studies, FBXL19 is degraded by the ubiquitin-proteasome system and its site-specific ubiquitination is mediated by SCFFBXW17 E3 ligase, which promotes cell migration.

Type-1 immunity and endogenous immune regulators predominate in the airway transcriptome during chronic lung allograft dysfunction.

Chronic lung allograft dysfunction (CLAD) remains the major complication limiting long-term survival among lung transplant recipients (LTRs). Limited understanding of CLAD immunopathogenesis and a paucity of biomarkers remain substantial barriers for earlier detection and therapeutic interventions for CLAD. We hypothesized the airway transcriptome would reflect key immunologic changes in disease. We compared airway brush-derived transcriptomic signatures in CLAD (n = 24) versus non-CLAD (n = 21) LTRs. A targeted assessment of the proteome using concomitant bronchoalveolar lavage (BAL) fluid for 24 cytokines/chemokines and alloimmune T cell responses was performed to validate the airway transcriptome. We observed an airway transcriptomic signature of differential genes expressed (DGEs) in CLAD marked by Type-1 immunity and striking upregulation of two endogenous immune regulators: indoleamine 2, 3 dioxygenase 1 (IDO-1) and tumor necrosis factor receptor superfamily 6B (TNFRSF6B). Advanced CLAD staging was associated with a more intense airway transcriptome signature. In a validation cohort using the identified signature, we found an area under the curve (AUC) of 0.77 for CLAD LTRs. Targeted proteomic analyses revealed a predominant Type-1 profile with detection of IFN-γ, TNF-α, and IL-1ß as dominant CLAD cytokines, correlating with the airway transcriptome. The airway transcriptome provides novel insights into CLAD immunopathogenesis and biomarkers that may impact diagnosis of CLAD.

Safety and Cumulative Incidence of Major Cardiovascular Events with Ticagrelor in Taiwanese Patients with Non-ST-Segment Elevation Myocardial Infarction: A 12-Month, Prospective, Phase IV, Multicenter, Single-Arm Study.

BACKGROUND: Ticagrelor, an oral, direct-acting, and reversible P2Y12 receptor antagonist, inhibits platelet activation and aggregation. This phase IV, single-arm study analyzed the safety and tolerability of ticagrelor in Taiwanese patients with non-ST-segment elevation myocardial infarction (NSTEMI) during 1 year of follow-up. METHODS: Patients aged ≥ 20 years with an index event of NSTEMI received ticagrelor (180 mg loading and 90 mg doses twice daily thereafter) plus low-dose aspirin (100 mg/day) for up to 1 year. Safety was evaluated according to adverse events (AEs), serious AEs (SAEs), and PLATO-defined bleeding events. The cumulative incidence of major cardiovascular (CV) events including CV death, myocardial infarction, and stroke was also evaluated. RESULTS: The safety population included 108 patients across 13 centers in Taiwan. During treatment, 32 (29.6%) patients had ≥ one PLATO-defined bleeding event. Major bleeding events occurred in seven (6.5%) patients with a Kaplan-Meier (KM) estimated event risk [95% confidence interval (CI)] of 7.1% (3.4%-14.4%), including life-threatening bleeding [four (3.7%) patients] and other major bleeding [three (2.8%) patients]. No PLATO-defined fatal bleeding was observed. SAEs were reported in 23 (21.3%) patients. Six (5.6%) patients experienced major CV events during the 1-year follow-up period, with a KM-estimated event risk (95% CI) of 5.6% (2.6%-12.0%). CONCLUSIONS: Ticagrelor for up to 1 year was associated with a low rate of major bleeding events and a low incidence of major CV events in Taiwanese patients with NSTEMI. The overall safety of ticagrelor was in accordance with the known safety profile of ticagrelor.

TRIM21 Mitigates Human Lung Microvascular Endothelial Cells' Inflammatory Responses to LPS.

Endothelial cell (EC) inflammation is regarded as an important pathogenic feature of many inflammatory diseases, including acute lung injury and sepsis. An increase in EC inflammation results in neutrophil infiltration from the blood to the site of inflammation, further promoting EC permeability. The ubiquitin E3 ligase TRIM21 has been implicated in human disorders; however, the roles of TRIM21 in endothelial dysfunction and acute lung injury have not been reported. Here, we reveal an antiinflammatory property of TRIM21 in a mouse model of acute lung injury and human lung microvascular ECs. Overexpression of TRIM21 by lentiviral vector infection effectively dampened LPS-induced neutrophil infiltration, cytokine release, and edema in mice. TRIM21 inhibited human lung microvascular endothelial cell inflammatory responses as evidenced by attenuation of the NF-κB pathway, release of IL-8, expression of intercellular adhesion molecules, and adhesion of monocytes to ECs. Furthermore, we demonstrated that TRIM21 was predominantly degraded by an increase in its monoubiquitination and lysosomal degradation after inflammatory stimuli. Thus, inhibition of vascular endothelial inflammation by TRIM21 provides a novel therapeutic target to lessen pulmonary inflammation.

Histone acetyltransferase CBP promotes function of SCF FBXL19 ubiquitin E3 ligase by acetylation and stabilization of its F-box protein subunit.

Ubiquitin E3 ligases mediate ubiquitination and degradation of intracellular proteins. We have shown that a relatively new Skp, Cullin, F-box (SCF) protein E3 ligase, SCF FBXL19, has an anti-inflammatory effect and controls actin cytoskeleton dynamics via targeting cell membrane receptor and small GTPases for their ubiquitination and degradation, but the molecular regulation of its subunit FBXL19 stability remains unclear. Here we show that FBXL19 degradation is controlled by the balance between its ubiquitination and acetylation. FBXL19 is an unstable protein with a half-life of ∼3 h. FBXL19 can be polyubiquitinated, and the proteasome inhibitor MG-132 prolongs FBXL19 half-life, suggesting that FBXL19 degradation is mediated in the ubiquitin-proteasome system. FBXL19 can also be acetylated, and enhancing acetylation of FBXL19 by a deacetylase inhibitor reduces FBXL19 ubiquitination levels. Acetylation-mimic FBXL19 mutant exhibits a longer half-life than wild type. An acetyltransferase CBP catalyzes acetylation of FBXL19. Inhibition or down-regulation of CBP reduces FBXL19 stability, whereas it is increased in CBP-overexpressing cells. Taken together, the data indicate that CBP-mediated acetylation reduces ubiquitination and stabilizes FBXL19. Further, we demonstrate that FBXL19 targets small GTPase Cdc42 for its ubiquitination and degradation, whereas this effect is reversed by inhibition of CBP, suggesting that CBP increases the effect of SCF FBXL19 E3 ligase through acetylation and stabilization of FBXL19. Our study reveals a new molecular model for regulation of SCF E3 ligase function by acetylation and stabilization of its subunit F-box protein.-Wei, J., Dong, S., Yao, K., Martinez, M. F. Y. M., Fleisher, P. R., Zhao, Y., Ma, H., Zhao, J. Histone acetyltransferase CBP promotes function of SCF FBXL19 ubiquitin E3 ligase by acetylation and stabilization of its F-box protein subunit.

SCFFBXO17 E3 ligase modulates inflammation by regulating proteasomal degradation of glycogen synthase kinase-3ß in lung epithelia.

Glycogen synthase kinase-3ß (GSK3ß) has diverse biological roles including effects on cellular differentiation, migration, and inflammation. GSK3ß phosphorylates proteins to generate phosphodegrons necessary for recognition by Skp1/Cullin-1/F-box (SCF) E3 ubiquitin ligases leading to subsequent proteasomal degradation of these substrates. However, little is known regarding how GSK3ß protein stability itself is regulated and how its stability may influence inflammation. Here we show that GSK3ß is degraded by the ubiquitin-proteasome pathway in murine lung epithelial cells through lysine 183 as an acceptor site for K48 polyubiquitination. We have identified FBXO17 as an F-box protein subunit that recognizes and mediates GSK3ß polyubiquitination. Both endogenous and ectopically expressed FBXO17 associate with GSK3ß, and its overexpression leads to decreased protein levels of GSK3ß. Silencing FBXO17 gene expression increased the half-life of GSK3ß in cells. Furthermore, overexpression of FBXO17 inhibits agonist-induced release of keratinocyte-derived cytokine (KC) and interleukin-6 (IL-6) production by cells. Thus, the SCFFBXO17 E3 ubiquitin ligase complex negatively regulates inflammation by targeting GSK3ß in lung epithelia.

Cross-talk between lysophosphatidic acid receptor 1 and tropomyosin receptor kinase A promotes lung epithelial cell migration.

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid, which plays a crucial role in the regulation of cell proliferation, migration, and differentiation. LPA exerts its biological effects mainly through binding to cell-surface LPA receptors (LPA1-6), which belong to the G protein-coupled receptor (GPCR) family. Recent studies suggest that cross-talk between receptor tyrosine kinases (RTKs) and GPCRs modulates GPCRs-mediated signaling. Tropomyosin receptor kinase A (TrkA) is a RTK, which mediates nerve growth factor (NGF)-induced biological functions including cell migration in neuronal and non-neuronal cells. Here, we show LPA1 transactivation of TrkA in murine lung epithelial cells (MLE12). LPA induced tyrosine phosphorylation of TrkA in both time- and dose-dependent manners. Down-regulation of LPA1 by siRNA transfection attenuated LPA-induced phosphorylation of TrkA, suggesting a cross-talk between LPA1 and TrkA. To investigate the molecular regulation of the cross-talk, we focused on the interaction between LPA1 and TrkA. We found that LPA induced interaction between LPA1 and TrkA. The LPA1/TrkA complex was localized on the plasma membrane and in the cytoplasm. The C-terminus of LPA1 was identified as the binding site for TrkA. Inhibition of TrkA attenuated LPA-induced phosphorylation of TrkA and LPA1 internalization, as well as lung epithelial cell migration. These studies provide a molecular mechanism for the transactivation of TrkA by LPA, and suggest that the cross-talk between LPA1 and TrkA regulates LPA-induced receptor internalization and lung epithelial cell migration.

Focal adhesion kinase-mediated activation of glycogen synthase kinase 3ß regulates IL-33 receptor internalization and IL-33 signaling.

IL-33, a relatively new member of the IL-1 cytokine family, plays a crucial role in allergic inflammation and acute lung injury. Long form ST2 (ST2L), the receptor for IL-33, is expressed on immune effector cells and lung epithelia and plays a critical role in triggering inflammation. We have previously shown that ST2L stability is regulated by the ubiquitin-proteasome system; however, its upstream internalization has not been studied. In this study, we demonstrate that glycogen synthase kinase 3ß (GSK3ß) regulates ST2L internalization and IL-33 signaling. IL-33 treatment induced ST2L internalization, and an effect was attenuated by inhibition or downregulation of GSK3ß. GSK3ß was found to interact with ST2L on serine residue 446 in response to IL-33 treatment. GSK3ß binding site mutant (ST2L(S446A)) and phosphorylation site mutant (ST2L(S442A)) are resistant to IL-33-induced ST2L internalization. We also found that IL-33 activated focal adhesion kinase (FAK). Inhibition of FAK impaired IL-33-induced GSK3ß activation and ST2L internalization. Furthermore, inhibition of ST2L internalization enhanced IL-33-induced cytokine release in lung epithelial cells. These results suggest that modulation of the ST2L internalization by FAK/GSK3ß might serve as a unique strategy to lessen pulmonary inflammation.

AM966, an Antagonist of Lysophosphatidic Acid Receptor 1, Increases Lung Microvascular Endothelial Permeability through Activation of Rho Signaling Pathway and Phosphorylation of VE-Cadherin.

Maintenance of pulmonary endothelial barrier integrity is important for reducing severity of lung injury. Lysophosphatidic acid (LPA) regulates cell motility, cytoskeletal rearrangement, and cell growth. Knockdown of LPA receptor 1 (LPA1) has been shown to mitigate lung injury and pulmonary fibrosis. AM966, an LPA1 antagonist exhibiting an antifibrotic property, has been considered to be a future antifibrotic medicine. Here, we report an unexpected effect of AM966, which increases lung endothelial barrier permeability. An electric cell-substrate sensing (ECIS) system was used to measure permeability in human lung microvascular endothelial cells (HLMVECs). AM966 decreased the transendothelial electrical resistance (TEER) value immediately in a dose-dependent manner. VE-cadherin and f-actin double immunostaining reveals that AM966 increases stress fibers and gap formation between endothelial cells. AM966 induced phosphorylation of myosin light chain (MLC) through activation of RhoA/Rho kinase pathway. Unlike LPA treatment, AM966 had no effect on phosphorylation of extracellular signal-regulated kinases (Erk). Further, in LPA1 silencing cells, we observed that AM966-increased lung endothelial permeability as well as phosphorylation of VE-cadherin and focal adhesion kinase (FAK) were attenuated. This study reveals that AM966 induces lung endothelial barrier dysfunction, which is regulated by LPA1-mediated activation of RhoA/MLC and phosphorylation of VE-cadherin.

[Generation and Regulation Technology of Stimulating Signals for Implantable Neural Stimulators].

The accuracy and robustness of stimulating signals are critical important for implantable neural stimulators since they stimulate neurons directly. The characteristics and applications of stimulating signals were depicted in the paper. The principles and features of five common generation and regulation technology of stimulating signals for implantable neural stimulators were introduced. The technical characteristics of them were analyzed. Finally, the development of the implantable neural simulators was prospected.

Serum starvation regulates E-cadherin upregulation via activation of c-Src in non-small-cell lung cancer A549 cells.

E-cadherin is essential for the integrity of adherens junctions between lung epithelial cells, and the loss of E-cadherin allows cell motility and is thought to promote lung cancer metastasis. While the downregulation of E-cadherin expression has been well characterized and is seen with transforming growth factor-ß1 (TGF-ß1) exposure, few studies have focused on E-cadherin upregulation. Here, we show that serum starvation causes increased E-cadherin expression via the activation of c-Src kinase in non-small-cell lung cancer A549 cells. Serum starvation increased E-cadherin protein levels in a time- and dose-dependent manner. E-cadherin mRNA transcripts were unchanged with starvation, while protein translation inhibition with cycloheximide attenuated E-cadherin protein induction by starvation, suggesting that E-cadherin is regulated at the translational level by serum starvation. c-Src is a nonreceptor tyrosine kinase known to regulate protein translation machinery; serum starvation caused early and sustained activation of c-Src in A549 cells followed by E-cadherin upregulation. Furthermore, overexpression of a dominant negative c-Src attenuated the induction of E-cadherin by serum deprivation. Finally, we observed that TGF-ß1 treatment attenuated the serum activation of c-Src as well as E-cadherin expression when cells were deprived of serum. In conclusion, our data demonstrate that the c-Src kinase is activated by serum starvation to increase E-cadherin expression in A549 cells, and these phenomena are antagonized by TGF-ß1. These novel observations implicate the c-Src kinase as an upstream inducer of E-cadherin protein translation with serum starvation and TGF-ß1 diametrically regulating c-Src kinase activity and thus E-cadherin abundance in A549 cells.

Overexpression of USP14 protease reduces I-κB protein levels and increases cytokine release in lung epithelial cells.

The ubiquitin-proteasome system is the major pathway of non-lysosomal intracellular protein degradation, playing an important role in a variety of cellular responses including cell division, proliferation, and apoptosis. Ubiquitin-specific protease 14 (USP14) is a component of proteasome regulatory subunit 19 S that regulates deubiquitinated proteins entering inside the proteasome core 20 S. The role of USP14 in protein degradation is still controversial. Several studies suggest that USP14 plays an inhibitory role in protein degradation. Here, in contrast, overexpression of USP14 induced I-κB degradation, which increased cytokine release in lung epithelial cells. Overexpression of HA-tagged USP14 (HA-USP14) reduced I-κB protein levels by increasing the I-κB degradation rate in mouse lung epithelial cells (MLE12). I-κB polyubiquitination was reduced in HA-USP14-overexpressed MLE12 cells, suggesting that USP14 regulates I-κB degradation by removing its ubiquitin chain, thus promoting the deubiquitinated I-κB degradation within the proteasome. Interestingly, we found that USP14 was associated with RelA, a binding partner of I-κB, suggesting that RelA is the linker between USP14 and I-κB. Lipopolysaccharide (LPS) treatment induced serine phosphorylation of USP14 as well as further reducing I-κB levels in HA-USP14-overexpressed MLE12 cells as compared with empty vector transfected cells. Further, overexpression of HA-USP14 increased the LPS-, TNFα-, or Escherichia coli-induced IL-8 release in human lung epithelial cells. This study suggests that USP14 removes the ubiquitin chain of I-κB, therefore inducing I-κB degradation and increasing cytokine release in lung epithelial cells.

A new mechanism of RhoA ubiquitination and degradation: roles of SCF(FBXL19) E3 ligase and Erk2.

RhoA is a small GTPase multifunctional protein that regulates cell proliferation and cytoskeletal reorganization. Regulation of its protein stability plays an important role in its biological functions. We have shown that a Skp1-Cul1-F-box (SCF) FBXL19 E3 ubiquitin ligase targets Rac1, a related member of the Rho family for ubiquitination and degradation. Here, SCF(FBXL19) mediates RhoA ubiquitination and proteasomal degradation in lung epithelial cells. Ectopically expressed FBXL19 decreased RhoA wild type, active, and inactive forms. Cellular depletion of FBXL19 increased RhoA protein levels and extended its half-life. FBXL19 bound the small GTPase in the cytoplasm leading to RhoA ubiquitination at Lys(135). A RhoA(K135R) mutant protein was resistant to SCF(FBXL19)-mediated ubiquitination and degradation and exhibited a longer lifespan. Protein kinase Erk2-mediated phosphorylation of RhoA was both sufficient and required for SCF(FBXL19)-mediated RhoA ubiquitination and degradation. Thus, SCF(FBXL19) targets RhoA for its disposal, a process regulated by Erk2. Ectopically expressed FBXL19 reduced phosphorylation of p27 and cell proliferation, a process mediated by RhoA. Further, FBXL19 cellular expression diminished lysophosphatidic acid (LPA)-induced phosphorylation of myosin light chain (MLC) and stress fiber formation. Hence, SCF(FBXL19) functions as a RhoA antagonist during cell proliferation and cytoskeleton rearrangement. These results provide the first evidence of an F-box protein targeting RhoA thereby modulating its cellular lifespan that impacts cell proliferation and cytoskeleton rearrangement.

F-box protein complex FBXL19 regulates TGFß1-induced E-cadherin down-regulation by mediating Rac3 ubiquitination and degradation.

BACKGROUND: Rac3 is a small GTPase multifunctional protein that regulates cell adhesion, migration, and differentiation. It has been considered as an oncogene in breast cancer; however, its role in esophageal cancer and the regulation of its stability have not been studied. F-box proteins are major subunits within the Skp1-Cullin-1-F-box (SCF) E3 ubiquitin ligases that recognize particular substrates for ubiquitination and proteasomal degradation. Recently, we have shown that SCFFBXL19 targets Rac1 and RhoA, thus regulating Rac1 and RhoA ubiquitination and degradation. Here, we demonstrate the role of FBXL19 in the regulation of Rac3 site-specific ubiquitination and stability. Expression of TGFß1 is associated with poor prognosis of esophageal cancer. TGFß1 reduces tumor suppressor, E-cadherin, expression in various epithelial-derived cancers. Here we investigate the role of FBXL19-mediated Rac3 degradation in TGFß1-induced E-cadherin down-regulation in esophageal cancer cells. METHODS: FBXL19-regulated endogenous and over-expressed Rac3 stability were determined by immunoblotting and co-immunoprecipitation. Esophageal cancer cells (OE19 and OE33) were used to investigate TGFß1-induced E-cadherin down-regulation by Immunoblotting and Immunostaining. RESULTS: Overexpression of FBXL19 decreased endogenous and over-expressed Rac3 expression by interacting and polyubiquitinating Rac3, while down-regulation of FBXL19 suppressed Rac3 degradation. Lysine166 within Rac3 was identified as an ubiquitination acceptor site. The FBXL19 variant with truncation at the N-terminus resulted in an increase in Rac3 degradation; however, the FBXL19 variant with truncation at the C-terminus lost its ability to interact with Rac3 and ubiquitinate Rac3 protein. Further, we found that Rac3 plays a critical role in TGFß1-induced E-cadherin down-regulation in esophageal cancer cells. Over-expression of FBXL19 attenuated TGFß1-induced E-cadherin down-regulation and esophageal cancer cells elongation phenotype. CONCLUSIONS: Collectively these data unveil that FBXL19 functions as an antagonist of Rac3 by regulating its stability and regulates the TGFß1-induced E-cadherin down-regulation. This study will provide a new potential therapeutic strategy to regulate TGFß1 signaling, thus suppressing esophageal tumorigenesis.

SCF E3 ligase F-box protein complex SCF(FBXL19) 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 serine(71) was essential for FBXL19-mediated Rac1 ubiquitination and depletion. Lysine(166) within Rac1 was identified as a polyubiquitination acceptor site. Rac1(S71A) and Rac1(K166R) 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 lysine(166) mutant-overexpressing cells. FBXL19 diminished formation of the migratory leading edge. Thus, SCF(FBXL19) 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.

The deubiquitinase USP40 preserves endothelial integrity by targeting the heat shock protein HSP90ß.

Endothelial cell (EC) barrier disruption and inflammation are the pathological hallmarks of vascular disorders and acute infectious diseases and related conditions, including the coronavirus disease 2019 (COVID-19) and sepsis. Ubiquitination plays a critical role in regulating the stability, intracellular trafficking, and enzymatic activity of proteins and is reversed by deubiquitinating enzymes (DUBs). The role of DUBs in endothelial biology is largely unknown. In this study, we report that USP40, a poorly characterized DUB, prevents EC barrier disruption through reductions in the activation of RhoA and phosphorylation of myosin light chain (MLC) and cofilin. Furthermore, USP40 reduces EC inflammation through the attenuation of NF-ĸB activation, ICAM1 expression, and leukocyte-EC adhesion. We further show that USP40 activity and expression are reduced in response to endotoxin challenge. Global depletion of USP40 and EC-targeted USP40 depletion in mice exacerbated experimental lung injury, whereas lentiviral gene transfer of USP40 protected against endotoxin-induced lung injury. Using an unbiased approach, we discovered that the protective effect of USP40 occurs through the targeting of heat shock protein 90ß (HSP90ß) for its deubiquitination and inactivation. Together, these data reveal a critical protective role of USP40 in vascular injury, identifying a unique mechanistic pathway that profoundly impacts endothelial function via DUBs.