Thalidomide Attenuates Mast Cell Activation by Upregulating SHP-1 Signaling and Interfering with the Action of CRBN.

Allergy is a chronic inflammatory disease, and its incidence has increased worldwide in recent years. Thalidomide, which was initially used as an anti-emetic drug but was withdrawn due to its teratogenic effects, is now used to treat blood cancers. Although the anti-inflammatory and immunomodulatory properties of thalidomide have been reported, little is known about its influence on the mast cell-mediated allergic reaction. In the present study, we aimed to evaluate the anti-allergic activity of thalidomide and the underlying mechanism using mouse bone marrow-derived mast cells (BMMCs) and passive cutaneous anaphylaxis (PCA) mouse models. Thalidomide markedly decreased the degranulation and release of lipid mediators and cytokines in IgE/Ag-stimulated BMMCs, with concurrent inhibition of FcεRI-mediated positive signaling pathways including Syk and activation of negative signaling pathways including AMP-activated protein kinase (AMPK) and SH2 tyrosine phosphatase-1 (SHP-1). The knockdown of AMPK or SHP-1 with specific siRNA diminished the inhibitory effects of thalidomide on BMMC activation. By contrast, the knockdown of cereblon (CRBN), which is the primary target protein of thalidomide, augmented the effects of thalidomide. Thalidomide reduced the interactions of CRBN with Syk and AMPK promoted by FcεRI crosslinking, thereby relieving the suppression of AMPK signaling and suppressing Syk signaling. Furthermore, oral thalidomide treatment suppressed the PCA reaction in mice. In conclusion, thalidomide suppresses FcεRI-mediated mast cell activation by activating the AMPK and SHP-1 pathways and antagonizing the action of CRBN, indicating that it is a potential anti-allergic agent.

AMPK is essential for IL-10 expression and for maintaining balance between inflammatory and cytoprotective signaling.

BACKGROUND: AMP-activated protein kinase (AMPK) exerts its anti-inflammatory effects by suppressing redox-sensitive nuclear factor kappa B (NF-κB) and pro-inflammatory cytokines including TNF-α. However, it is unclear whether AMPK regulates anti-inflammatory cytokine expressions in the presence of oxidative stress-induced inflammation. We sought to elucidate the mechanisms whereby AMPK regulates inflammatory cytokine expressions under NADPH oxidase (NOX)-induced oxidative stress. METHODS: HT-29 human colonic epithelial cells transfected with AMPKα shRNA and mouse models with AMPKα knocked out in epithelial cells (AMPKαfl/fl-Vil-Cre) or macrophages (AMPKαfl/fl-Lyz2-Cre) were used to examine the effects of AMPK and NOX on signaling pathways and cytokine expressions. RESULTS: In HT-29 cells, 5-hydroxytryptamine (5-HT)-induced NOX activity was enhanced by AMPKα silencing, and resulted in inflammatory cell death. AMPKα deletion specific for colon epithelial cells (AMPKαfl/fl-Vil-Cre) or macrophages (AMPKαfl/fl-Lyz2-Cre) intensified 5-HT- or dextran sulfate sodium (DSS)-induced upregulations of NOX2, TNF-α, and IL-6, but completely abolished basal and 5-HT- or DSS-induced upregulation of IL-10 in colon epithelium. Furthermore, 5-HT- and DSS-induced changes were accompanied by marked upregulations of increased inflammatory signaling pathways linked to NF-κB, AP-1, and STAT3 transcription factors, and to GATA, a cell fate-directing signaling. In addition, AMPKα deletion significantly fortified 5-HT- or DSS-induced downregulations of cytoprotective signaling pathways (Nrf2, HIF-1α, and KLF4). CONCLUSION: Basal AMPKα maintains an anti-inflammatory state by inhibiting NOX, balancing pro-/anti-inflammatory signaling pathways, and directing IL-10 production. When these regulatory roles of AMPK are diminished by oxidative stress, colon epithelium undergoes inflammation despite IL-10 production.

Author Correction: Sirt1 negatively regulates FcεRI-mediated mast cell activation through AMPK- and PTP1B-dependent processes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A common signaling pathway leading to degranulation in mast cells and its regulation by CCR1-ligand.

BACKGROUND: Signal transduction pathways mediated by various receptors expressed on mast cells are thought to be complex, and inhibitory signals that turn off activating signals are not known. METHODS: Upstream signaling cascades mediated by several known receptors in bone marrow-derived mast cells that lead to degranulation and mediator release were studied by immunoblotting and immunoprecipitation. Small interfering RNAs and knockout mice were used to confirm findings. RESULTS: All ligands tested including IgE/Ag, SCF, HSP70, CCL3, and its valiant eMIP induced phosphorylation of linker for activation of T cells (LAT), which triggered their receptor-mediated downstream signaling cascades that controlled degranulation and mediator release. Phosphorylation of lymphocyte-specific protein kinase (Lck) was induced by each ligand, which commonly played an indispensable role in LAT phosphorylation. In contrast, phosphorylation of spleen tyrosine kinase was additionally induced in cells stimulated only with IgE/Ag and SCF, which is also associated with LAT phosphorylation in part. Degranulation and mediator release induced by IgE/Ag, SCF, or HSP70 were enhanced by nanomolar doses of CCR1 ligands CCL3 and eMIP via enhanced LAT phosphorylation. On the other hand, micromolar doses of CCR1 ligand inhibited degranulation and mediator release from mast cells stimulated with IgE/Ag, SCF, or HSP70 by de-phosphorylation of phosphorylated Lck with Src homology region 2 domain-containing phosphatase-1. CONCLUSIONS: Linker for activation of T cells plays a central role in signal transduction pathways in mast cells stimulated with any ligand tested. Dose-dependent alternate costimulation and inhibition of CCR1 ligands in IgE/Ag-, SCF-, or HSP70-stimulated mast cells occur at the level of Lck-LAT phosphorylation.

Structural insights into stressosome assembly.

The stressosome transduces environmental stress signals to SigB to upregulate SigB-dependent transcription, which is required for bacterial viability. The stressosome core is composed of RsbS and at least one of the RsbR paralogs. A previous cryo-electron microscopy (cryo-EM) structure of the RsbRA-RsbS complex determined under a D2 symmetry restraint showed that the stressosome core forms a pseudo-icosahedron consisting of 60 STAS domains of RsbRA and RsbS. However, it is still unclear how RsbS and one of the RsbR paralogs assemble into the stressosome. Here, an assembly model of the stressosome is presented based on the crystal structure of the RsbS icosahedron and cryo-EM structures of the RsbRA-RsbS complex determined under diverse symmetry restraints (nonsymmetric C1, dihedral D2 and icosahedral I envelopes). 60 monomers of the crystal structure of RsbS fitted well into the I-restrained cryo-EM structure determined at 4.1 Šresolution, even though the STAS domains in the I envelope were averaged. This indicates that RsbS and RsbRA share a highly conserved STAS fold. 22 protrusions observed in the C1 envelope, corresponding to dimers of the RsbRA N-domain, allowed the STAS domains of RsbRA and RsbS to be distinguished in the stressosome core. Based on these, the model of the stressosome core was reconstructed. The mutation of RsbRA residues at the binding interface in the model (R189A/Q191A) significantly reduced the interaction between RsbRA and RsbS. These results suggest that nonconserved residues in the conserved STAS folds between RsbS and RsbR paralogs determine stressosome assembly.

Sauchinone suppresses FcεRI-mediated mast cell signaling and anaphylaxis through regulation of LKB1/AMPK axis and SHP-1-Syk signaling module.

Sauchinone, the biologically active lignan of Saururus chinensis, has been reported to have anti-inflammatory, antitumor, antioxidant, and hepatoprotective properties. However, little is known about the effect of sauchinone on FcεRI-mediated mast cell activation. The aim of this study was to evaluate the anti-allergic activity of sauchinone and the underlying mechanism using mouse bone marrow-derived mast cells (BMMCs) and the mast cell-mediated passive cutaneous anaphylaxis (PCA) model. Sauchinone markedly suppressed FcεRI-mediated activation of positive signaling mediators, including Syk, linker for activation of T cells (LAT), phospholipase C (PLC)γ, mitogen-activated protein (MAP) kinases, Akt, IκB kinase (IKK), and intracellular Ca2+, and increased the activation of negative signaling mediators, including liver kinase B (LKB)1/AMP-activated protein kinase (AMPK) and Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1. Interestingly, sauchinone increased the interaction between SHP-1 and Syk. Consequently, sauchinone significantly suppressed FcεRI-mediated BMMC degranulation and synthesis of eicosanoids and cytokines. These inhibitory effects of sauchinone were diminished in BMMCs treated with siRNAs targeting LKB1, AMPKα2, or SHP-1, and in BMMCs isolated from AMPKα2-deficient mice. In addition, administration of sauchinone markedly suppressed the IgE-mediated PCA reaction in wild-type mice, and this inhibitory effect was significantly reduced in AMPKα2-/- mice. Taken together, these data suggest that sauchinone suppresses FcεRI-mediated mast cell activation and anaphylaxis through modulation of the LKB1/AMPK and SHP-1/Syk pathways. Therefore, sauchinone might be a potential therapeutic agent for the treatment of allergic inflammatory diseases.

Activation of Mevalonate Pathway via LKB1 Is Essential for Stability of Treg Cells.

The function of regulatory T (Treg) cells depends on lipid oxidation. However, the molecular mechanism by which Treg cells maintain lipid metabolism after activation remains elusive. Liver kinase B1 (LKB1) acts as a coordinator by linking cellular metabolism to substrate AMP-activated protein kinase (AMPK). We show that deletion of LKB1 in Treg cells exhibited reduced suppressive activity and developed fatal autoimmune inflammation. Mechanistically, LKB1 induced activation of the mevalonate pathway by upregulating mevalonate genes, which was essential for Treg cell functional competency and stability by inducing Treg cell proliferation and suppressing interferon-gamma and interleukin-17A expression independently of AMPK. Furthermore, LKB1 was found to regulate intracellular cholesterol homeostasis and to promote the mevalonate pathway. In agreement, mevalonate and its metabolite geranylgeranyl pyrophosphate inhibited conversion of Treg cells and enhanced survival of LKB1-deficient Treg mice. Thus, LKB1 is a key regulator of lipid metabolism in Treg cells, involved in optimal programming of suppressive activity, immune homeostasis, and tolerance.

Ovarian tumor domain-containing ubiquitin aldehyde binding protein 1 inhibits inflammation by regulating Nur77 stability.

Nur77 (NR4A1) plays an important role in various inflammatory responses. Nur77 is rapidly degraded in cells and its protein level is critically controlled. Although few E3 ligases regulating the Nur77 protein have been defined, the deubiquitinase (DUB) responsible for Nur77 stability has not been reported to date. We identified ovarian tumor domain-containing ubiquitin aldehyde binding protein 1 (OTUB1) as a DUB that stabilizes Nur77 by preventing its proteasomal degradation. We found that OTUB1 interacted with Nur77 to deubiquitinate it, thereby stabilizing Nur77 in an Asp88-dependent manner. This suggests that OTUB1 targets Nur77 for deubiquitination via a non-canonical mechanism. Functionally, OTUB1 inhibited TNFα-induced IL-6 production by promoting Nur77 protein stability. OTUB1 modulated the stability of Nur77 as a counterpart of tripartite motif 13 (Trim13). That is, OTUB1 reduced the ubiquitination and degradation of Nur77 potentiated by Trim13. In addition, this DUB also inhibited IL-6 production, which was further amplified by Trim13 in TNFα-induced responses. These findings suggest that OTUB1 is an important regulator of Nur77 stability and plays a role in controlling the inflammatory response.

AMPK is down-regulated by the CRL4A-CRBN axis through the polyubiquitination of AMPKα isoforms.

As a master regulator for metabolic and energy homeostasis, AMPK controls the activity of metabolic enzymes and transcription factors in response to cellular ATP status. AMPK has been thus recognized as a main target for the regulation of cellular energy metabolism. Here, we report that AMPK can be down-regulated by the cullin-RING ubiquitin E3 ligase 4A (CRL4A) with cereblon (CRBN). CRL4A interacted with AMPK holoenzymes and mediated AMPKα-specific polyubiquitination for its proteasomal degradation through non-K48 polyubiquitin linkages. In the ubiquitination system, CRBN was required for efficient polyubiquitination of AMPKα subunits. Consistently, polyubiquitination of AMPKα subunits was reduced by inhibitors of CRL4A-CRBN. Physiologic function of AMPK down-regulation by CRL4-CRBN was also confirmed using mouse bone marrow-derived mast cells (BMMCs). The inactivation of CRL4A-CRBN in BMMC increased AMPK stability and suppressed secretion of allergic mediators via AMPK activation followed by MAPK inhibition. In addition, CRBN knockout of BMMC also decreased allergic responses in mice. Our results suggest that the CRL4A-CRBN axis could be a target for the regulation of AMPK-dependent responses.-Kwon, E., Li, X., Deng, Y., Chang, H. W., Kim, D. Y. AMPK is down-regulated by the CRL4A-CRBN axis through the polyubiquitination of AMPKα isoforms.

Esculentoside B inhibits inflammatory response through JNK and downstream NF-κB signaling pathway in LPS-triggered murine macrophage RAW 264.7 cells.

Natural compound esculentoside B (EsB), (2S,4aR,6aR,6aS,6bR,8aR,9R,10R,11S,12aR,14bS)-11-hydroxy-9-(hydroxymethyl)-2 methoxycarbonyl-2,6a,6b,9,12a-pentamethyl-10-[(2S,3R,4S,5R)-3,4,5-trihydroxyoxan-2-yl]oxy-1,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydropicene-4a-carboxylic acid with molecular weight of 664.833, isolated from roots of Phytolacca acinosa Roxb has been widely used as a constituent of traditional Chinese medicine (TCM). However, the anti-inflammatory capacity of EsB has not been reported yet. Therefore, the objective of this study was to investigate anti-inflammatory activities of EsB in LPS-treated macrophage RAW 264.7 cells. EsB could inhibit nitric oxide (NO) production. EsB also suppressed gene and protein expression levels of inducible isoform of NO synthase (NOS) and cyclooxygenase-2 in a dose-dependent manner. In addition, EsB decreased gene expression and protein secretion levels of pro-inflammatory cytokines such as IL-1ß, TNF-α, and IL-6. EsB remarkably suppressed nuclear translocation of nuclear factor kappa-B (NF-κB) from cytosolic space. Phosphorylation of IκB was also inhibited by EsB. Moreover, EsB specifically down-regulated phospho-c-Jun N-terminal kinase (p-JNK), but not p-p38 or phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2). Taken together, these results suggest that EsB has inhibitory effect on inflammatory response by inactivating NF-κB and p-JNK. It could be used as a new modulatory drug for effective treatment of inflammation-related diseases.

Esculentoside H inhibits colon cancer cell migration and growth through suppression of MMP-9 gene expression via NF-kB signaling pathway.

A water-soluble saponin, Esculentoside H (EsH), 3-O-(O-ß-d-glucopyranosyl-(1→4)-ß-d-xylopyranosyl)-28-ß-d-glucopyranosylphytolaccagenin has been isolated and purified from the root extract of perennial plant Phytolacca esculenta. EsH is known to be an anticancer compound, having a capacity for TNF-α release. However, the effects of EsH on migration and growth in tumor cells have not yet been reported. In the current study, the suppressive effects of EsH on phorbol 12-myristate 13-acetate (PMA)-induced cell migration were examined in murine colon cancer CT26 cells and human colon cancer HCT116 cells. Interestingly, the transwell assay and wound healing show that EsH suppresses the PMA-induced migration and growth potential of HCT116 and CT26 colon cancer cells, respectively. EsH dose-dependently suppressed matrix metalloproteinases-9 (MMP-9) expression that was upregulated upon PMA treatment in messenger RNA levels and protein secretion. Since the expression of MMP-9 is correlated with nuclear factor-κB (NF-κB) signaling, it has been examined whether EsH inhibits PMA-induced IκB phosphorylation that leads to the suppression of NK-κB nuclear translocation. EsH repressed the phosphorylation level of JNK, but not extracellular signal-regulated kinase and p38 signaling when the cells were treated with PMA. Overall, these results demonstrated that EsH could suppress cancer migration through blockage of the JNK1/2 and NF-κB signaling-mediated MMP-9 expression.

The orphan nuclear receptor NR4A1 promotes FcεRI-stimulated mast cell activation and anaphylaxis by counteracting the inhibitory LKB1/AMPK axis.

BACKGROUND: Nuclear receptor subfamily 4 group A member 1 (NR4A1), an orphan nuclear receptor, has been implicated in several biological events such as metabolism, apoptosis, and inflammation. Recent studies indicate a potential role for NR4A1 in mast cells, yet its role in allergic responses remains largely unknown. OBJECTIVES: The aim of this study was to clarify the role of NR4A1 in mast cell activation and anaphylaxis. METHODS: To evaluate the function of NR4A1 in mast cells, the impacts of siRNA knockdown, gene knockout, adenoviral overexpression, and pharmacological inhibition of NR4A1 on FcεRI signaling and effector functions in mouse bone marrow-derived mast cells (BMMCs) in vitro and on anaphylactic responses in vivo were evaluated. RESULTS: Knockdown or knockout of NR4A1 markedly suppressed degranulation and lipid mediator production by FcεRI-crosslinked BMMCs, while its overexpression augmented these responses. Treatment with a NR4A1 antagonist also blocked mast cell activation to a similar extent as NR4A1 knockdown or knockout. Moreover, mast cell-specific NR4A1-deficient mice displayed dampened anaphylactic responses in vivo. Mechanistically, NR4A1 promoted FcεRI signaling by counteracting the liver kinase B1 (LKB1)/adenosine monophosphate-activated protein kinase (AMPK) axis. Following FcεRI crosslinking, NR4A1 bound to the LKB1/AMPK complex and sequestered it in the nucleus, thereby promoting FcεRI downstream signaling pathways. Silencing or knockout of LKB1/AMPK largely abrogated the effect of NR4A1 on mast cell activation. Additionally, NR4A1 facilitated spleen tyrosine kinase activation independently of LKB1/AMPK. CONCLUSIONS: Nuclear receptor subfamily 4 group A member 1 positively regulates mast cell activation by antagonizing the LKB1-AMPK-dependent negative regulatory axis. This finding may provide a novel therapeutic strategy for the development of anti-allergic compounds.

Seongsanamides A-D: Antiallergic Bicyclic Peptides from Bacillus safensis KCTC 12796BP.

Six seongsanamides were isolated from the culture broth of Bacillus safensis KCTC 12796BP, and their structures were elucidated by spectroscopic data analysis combined with Marfey's method, electronic circular dichroism calculations, and biosynthetic gene cluster analysis. Compounds 1-4 were bicyclic peptides with isodityrosine residues; 5 and 6 were monocyclic peptides. Only the bicyclic seongsanamides inhibited degranulation and LTC4/PGD2 generation in IgE/Ag-stimulated bone marrow-derived mast cells. Oral administration of 1 suppressed mast cell-dependent passive cutaneous anaphylaxis reaction.

The E3 ubiquitin ligase Trim13 regulates Nur77 stability via casein kinase 2α.

Nur77 is a member of the NR4A subfamily of nuclear receptors and has been shown to regulate various biological processes such as apoptosis and inflammation. Here, we show that Nur77 ubiquitination is mediated by the tripartite motif 13 (Trim13), a RING-type E3 ubiquitin ligase. The interaction between Nur77 and Trim13 was confirmed by co-immunoprecipitation. Moreover, we found that Lys539 in Nur77 ubiquitination is targeted for Trim13, which leads to Nur77 degradation. The Trim13-mediated ubiquitination of Nur77 was optimal in the presence of the E2 enzyme UbcH5. Importantly, in addition to Trim13-mediated ubiquitination, the stability of Nur77 was also regulated by casein kinase 2α (CK2α). Pharmacological inhibition of CK2 markedly increased Nur77 levels, whereas overexpression of CK2α, but not its inactive mutant, dramatically decreased Nur77 levels by promoting Nur77 ubiquitination. CK2α phosphorylated Ser154 in Nur77 and thereby regulated Nur77 protein levels by promoting its ubiquitin-mediated degradation. Importantly, we also show that degradation of Nur77 is involved in TNFα-mediated IL-6 production via CK2α and Trim13. Taken together, these results suggest that the sequential phosphorylation and ubiquitination of Nur77 controls its degradation, and provide a therapeutic approach for regulating Nur77 activity through the CK2α-Trim13 axis as a mechanism to control the inflammatory response.

Tanshinone IIA suppresses FcεRI-mediated mast cell signaling and anaphylaxis by activation of the Sirt1/LKB1/AMPK pathway.

AMP-activated protein kinase (AMPK) and its upstream mediators liver kinase B1 (LKB1) and sirtuin 1 (Sirt1) are generally known as key regulators of metabolism. We have recently reported that the AMPK pathway negatively regulates mast cell activation and anaphylaxis. Tanshinone IIA (Tan IIA), an active component of Salvia miltiorrhiza extract that is currently used for the treatment of cardiovascular and cerebrovascular diseases, shows anti-diabetic activity and improves insulin resistance in db/db mice through activation of AMPK. The aim of this study was to evaluate the anti-allergic activity of Tan IIA in vivo and to investigate the underlying mechanism in vitro in the context of AMPK signaling. The anti-allergic effect of Tan IIA was evaluated using mouse bone marrow-derived mast cells (BMMCs) from AMPKα2-/- or Sirt1-/- mice, or BMMCs transfected with siRNAs specific for AMPKα2, LKB1, or Sirt1. AMPKα2-/- and Sirt1-/- mice were used to confirm the anti-allergic effect of Tan IIA in anaphylaxis in vivo. Tan IIA dose-dependently inhibited FcεRI-mediated degranulation and production of eicosanoids and cytokines in BMMCs. These inhibitory effects were diminished by siRNA-mediated knockdown or genetic deletion of AMPKα2 or Sirt1. Moreover, Tan IIA inhibited a mast cell-mediated local passive anaphylactic reaction in wild-type mice, but not in AMPKα2-/- or Sirt1-/- mice. In conclusion, Tan IIA suppresses FcεRI-mediated mast cell activation and anaphylaxis through activation of the inhibitory Sirt1-LKB1-AMPK pathway. Thus, Tan IIA may be useful as a new therapeutic agent for mast cell-mediated allergic diseases.

Induction of GD3/α1-adrenergic receptor/transglutaminase 2-mediated erythroid differentiation in chronic myelogenous leukemic K562 cells.

The disialic acid-containing glycosphingolipid GD3 recruited membrane transglutaminase 2 (TG2) as a signaling molecule for erythroid differentiation in human chronic myelogenous leukemia (CML) K562 cells. The α1-adrenergic receptor (α1-AR)/TG2-mediated signaling pathway regulated GD3 functions, including gene expression and production, to differentiate CML K562 cells into erythroid lineage cells. Epinephrine, an AR agonist, increased membrane recruitment as well as GTP-photoaffinity of TG2, inducing GD3 synthase gene expression. Epinephrine activated PI3K/Akt signaling and GTPase downstream of TG2 activated Akt. The coupling of TG2 and GD3 production was specifically suppressed by prazosin (α1-AR antagonist), but not by propranolol (ß-AR antagonist) or rauwolscine (α2-AR antagonist), indicating α1-AR specificity. Small interfering RNA (siRNA) experiment results indicated that the α1-AR/TG2-mediated signaling pathway activated PKCs α and δ to induce GD3 synthase gene expression. Transcription factors CREB, AP-1, and NF-κB regulated GD3 synthase gene expression during α1-AR-induced differentiation in CML K562 cells. In addition, GD3 synthase gene expression was upregulated in TG2-transfected cells via α1-AR with expression of erythroid lineage markers and benzidine-positive staining. α1-AR/TG2 signaling pathway-directed GD3 production is a crucial step in erythroid differentiation of K562 cells and GD3 interacts with α1-AR/TG2, inducing GD3/α1-AR/TG2-mediated erythroid differentiation. These results suggest that GD3, which acts as a membrane mediator of erythroid differentiation in CML cells, provides a therapeutic avenue for leukemia treatment.

Isothiocyanates suppress the invasion and metastasis of tumors by targeting FAK/MMP-9 activity.

Isothiocyanates, which are present as glucosinolate precursors in cruciferous vegetables, have strong activity against various cancers. Here, we compared the anti-metastatic effects of isothiocyanates (benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC), and sulforaphane (SFN)) by examining how they regulate MMP-9 expression. Isothiocyanates, particularly PEITC, suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MMP-9 activity and invasion in various cancer cell lines. By contrast, N-methyl phenethylamine, a PEITC analog without an isothiocyanate functional group, had no effect. A reporter gene assay demonstrated that BITC, PEITC, and SFN suppressed TAP-induced MMP-9 expression by inhibiting AP-1 and NF-κB in U20S osteosarcoma cells. All three compounds reduced phosphorylation of FAK, ERK1/2, and Akt. In addition, MMP-9 expression was downregulated by inhibiting FAK, ERK1/2, and Akt. Isothiocyanates-mediated inhibition of FAK phosphorylation suppressed phosphorylation of ERK1/2 and Akt in U2OS and A549 cells, along with the translocation of p65 and c-Fos, suggesting that isothiocyanates inhibit MMP-9 expression and cell invasion by blocking phosphorylation of FAK. Furthermore, isothiocyanates, abolished MMP-9 expression and tumor metastasis in vivo with the following efficacy: PEITC>BITC>SFN. Thus, isothiocyanates act as anti-metastatic compounds that suppress MMP-9 activity/expression by inhibiting NF-κB and AP-1 via suppression of the FAK/ERK and FAK/Akt signaling pathways.

Crystal structure of mimivirus uracil-DNA glycosylase.

Cytosine deamination induced by stresses or enzymatic catalysis converts deoxycytidine into deoxyuridine, thereby introducing a G to A mutation after DNA replication. Base-excision repair to correct uracil to cytosine is initiated by uracil-DNA glycosylase (UDG), which recognizes and eliminates uracil from DNA. Mimivirus, one of the largest known viruses, also encodes a distinctive UDG gene containing a long N-terminal domain (N-domain; residues 1-130) and a motif-I (residues 327-343), in addition to the canonical catalytic domain of family I UDGs (also called UNGs). To understand the structural and functional features of the additional segments, we have determined the crystal structure of UNG from Acanthamoeba polyphaga mimivirus (mvUNG). In the crystal structure of mvUNG, residues 95-130 in the N-domain bind to a hydrophobic groove in the catalytic domain, and motif-I forms a short ß-sheet with a positively charged surface near the active site. Circular dichroism spectra showed that residues 1-94 are in a random coil conformation. Deletion of the three additional fragments reduced the activity and thermal stability, compared to full-length mvUNG. The results suggested that the mvUNG N-domain and motif-I are required for its structural and functional integrity.

Sirt1 negatively regulates FcεRI-mediated mast cell activation through AMPK- and PTP1B-dependent processes.

Sirt1, a key regulator of metabolism and longevity, has recently been implicated in the regulation of allergic reactions, although the underlying mechanism remains unclear. Here we show that Sirt1 negatively regulates FcεRI-stimulated mast cell activation and anaphylaxis through two mutually regulated pathways involving AMP-activated protein kinase (AMPK) and protein tyrosine phosphatase 1B (PTP1B). Mast cell-specific knockout of Sirt1 dampened AMPK-dependent suppression of FcεRI signaling, thereby augmenting mast cell activation both in vitro and in vivo. Sirt1 inhibition of FcεRI signaling also involved an alternative component, PTP1B, which attenuated the inhibitory AMPK pathway and conversely enhanced the stimulatory Syk pathway, uncovering a novel role of this phosphatase. Moreover, a Sirt1 activator resveratrol stimulated the inhibitory AMPK axis, with reciprocal suppression of the stimulatory PTP1B/Syk axis, thus potently inhibiting anaphylaxis. Overall, our results provide a molecular explanation for the beneficial role of Sirt1 in allergy and underscore a potential application of Sirt1 activators as a new class of anti-allergic agents.

Structural insights into the regulation of Bacillus subtilis SigW activity by anti-sigma RsiW.

Bacillus subtilis SigW is localized to the cell membrane and is inactivated by the tight interaction with anti-sigma RsiW under normal growth conditions. Whereas SigW is discharged from RsiW binding and thus initiates the transcription of its regulon under diverse stress conditions such as antibiotics and alkaline shock. The release and activation of SigW in response to extracytoplasmic signals is induced by the regulated intramembrane proteolysis of RsiW. As a ZAS (Zinc-containing anti-sigma) family protein, RsiW has a CHCC zinc binding motif, which implies that its anti-sigma activity may be regulated by the state of zinc coordination in addition to the proteolytic cleavage of RsiW. To understand the regulation mode of SigW activity by RsiW, we determined the crystal structures of SigW in complex with the cytoplasmic domain of RsiW, and compared the conformation of the CHCC motif in the reduced/zinc binding and the oxidized states. The structures revealed that RsiW inhibits the promoter binding of SigW by interacting with the surface groove of SigW. The interaction between SigW and RsiW is not disrupted by the oxidation of the CHCC motif in RsiW, suggesting that SigW activity might not be regulated by the zinc coordination states of the CHCC motif.