OTUB1/NDUFS2 axis promotes pancreatic tumorigenesis through protecting against mitochondrial cell death.

Pancreatic cancer is one of the most fatal cancers in the world. A growing number of studies have begun to demonstrate that mitochondria play a key role in tumorigenesis. Our previous study reveals that NDUFS2 (NADH: ubiquinone oxidoreductase core subunit S2), a core subunit of the mitochondrial respiratory chain complex I, is upregulated in Pancreatic adenocarcinoma (PAAD). However, its role in the development of PAAD remains unknown. Here, we showed that NDUFS2 played a critical role in the survival, proliferation and migration of pancreatic cancer cells by inhibiting mitochondrial cell death. Additionally, protein mass spectrometry indicated that the NDUFS2 was interacted with a deubiquitinase, OTUB1. Overexpression of OTUB1 increased NDUFS2 expression at the protein level, while knockdown of OTUB1 restored the effects in vitro. Accordingly, overexpression and knockdown of OTUB1 phenocopied those of NDUFS2 in pancreatic cancer cells, respectively. Mechanically, NDUFS2 was deubiquitinated by OTUB1 via K48-linked polyubiquitin chains, resulted in an elevated protein stability of NDUFS2. Moreover, the growth of OTUB1-overexpressed pancreatic cancer xenograft tumor was promoted in vivo, while the OTUB1-silenced pancreatic cancer xenograft tumor was inhibited in vivo. In conclusion, we revealed that OTUB1 increased the stability of NDUFS2 in PAAD by deubiquitylation and this axis plays a pivotal role in pancreatic cancer tumorigenesis and development.

Amplified Targeted Drug Delivery Independent of Target Number through Alternative Administration of Two Matched Nanoparticles.

Targeting nanoparticles (NPs) based on the specific binding of ligands with molecular targets provides a promising tool for tissue-selective drug delivery. However, the number of molecular targets on the cell surface is limited, hindering the number of NPs that can bind and, thus, limiting the therapeutic outcome. Although several strategies have been developed to enhance drug delivery, such as enhancing drug loading and circulation time or increasing the enhanced permeability and retention effect of nanocarriers, none have resolved this issue. Herein, we designed a simple method for amplified and targeted drug delivery using two matched NPs. One NP was aptamer-functionalized to specifically bind to target cells, while the other was aptamer-complementary DNA-functionalized to specifically bind to aptamer-NPs. Alternate administration of the two matched NPs enables their continuous accumulation in the disease site despite their limited molecular targets. As a proof of concept, the method was tested in a breast cancer model and significantly enhanced chemotherapy of tumor cells in vitro and in vivo. The potential applications of this method in a brain injury model were also demonstrated. Overall, the study describes a method for amplified targeted drug delivery independent of the target number.

Bullatine A exerts anti-inflammatory effects by inhibiting the ROS/JNK/NF-κB pathway and attenuating systemic inflammatory responses in mice.

CONTEXT: Aconiti brachypodi Radix (Xue-shang-yi-zhi-hao) is a traditional Chinese herbal medicine that is capable of anti-analgesic and anti-inflammatory effects. Bullatine A (BA) is one of the major active ingredients of this plant, and most of the previous studies reported that it has anti-analgesic effects. However, the mechanism of BA anti-inflammatory remains unclear. OBJECTIVE: This study investigates the anti-inflammatory activities of BA, both in vitro and in vivo, and elucidates its mechanism. MATERIALS AND METHODS: In vitro, BA (10, 20, 40 and 80 µM) was added to 1 µg/mL of lipopolysaccharide (LPS)-activated microglia BV2 cells and immortalized murine bone marrow-derived macrophages, respectively. After 6 h, the mRNA and protein levels of inflammatory factors were determined by real-time quantitative PCR and western blotting. In vivo, C57BL/6 mice were randomly divided into control, model (5 mg/kg dose of LPS) and treated groups (LPS with 5, 10 or 20 mg/kg dose of BA) to evaluate the anti-inflammatory efficacy of BA. RESULTS: BA significantly inhibited LPS-induced expression of inflammatory factors, such as IL-1ß, IL-6, TNF-α, inducible nitric oxide synthase (iNOS) and COX-2. Further investigations showed that BA reduced the translocation of NF-κB p65 (38.5%, p < 0.01). BA also reduced the phosphorylation of c-Jun N-terminal kinase (JNK) (11.2%, p < 0.05) and reactive oxygen species (ROS) generation (24.2%, p < 0.01). Furthermore, BA treatment attenuated the LPS-primed inflammatory response and liver and lung damage in vivo. CONCLUSIONS: BA can inhibit the inflammatory response in part through the ROS/JNK/NF-κB signalling pathway, providing a theoretical basis for the clinical application of BA in the treatment of periphery inflammatory diseases.

The XPO1 Inhibitor KPT-8602 Ameliorates Parkinson's Disease by Inhibiting the NF-κB/NLRP3 Pathway.

Exportin 1 (XPO1) is an important transport receptor that mediates the nuclear export of various proteins and RNA. KPT-8602 is a second-generation inhibitor of XPO1, demonstrating the lowest level of side effects, and is currently in clinical trials for the treatment of cancers. Previous studies suggest that several first-generation inhibitors of XPO1 demonstrate anti-inflammation activities, indicating the application of this drug in inflammation-related diseases. In this study, our results suggested the potent anti-inflammatory effect of KPT-8602 in vitro and in vivo. KPT-8602 inhibited the activation of the NF-κB pathway by blocking the phosphorylation and degradation of IκBα, and the priming of NLRP3. Importantly, the administration of KPT-8602 attenuated both lipopolysaccharide (LPS)-induced peripheral inflammation and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neuroinflammation in vivo. In addition, the tissue damage was also ameliorated by KPT-8602, indicating that KPT-8602 could be used as a novel potential therapeutic agent for the treatment of inflammasome-related diseases such as Parkinson's disease, through the regulation of the NF-κB signaling pathway and the NLRP3 inflammasome.

1,2,4-Trimethoxybenzene selectively inhibits NLRP3 inflammasome activation and attenuates experimental autoimmune encephalomyelitis.

NOD-like receptor (NLR) family pyrin domain-containing-3 (NLRP3) inflammasome is implicated in inflammation-associated diseases such as multiple sclerosis, Parkinson's disease, and stroke. Targeting the NLRP3 inflammasome is beneficial to these diseases, but few NLRP3 inflammasome-selective inhibitors are identified to date. Essential oils (EOs) are liquid mixtures of volatile and low molecular-weight organic compounds extracted from aromatic plants, which show various pharmacological activities, including antibacterial, antifungal, antiviral, antioxidant, and anti-inflammatory properties. In this study we screened active ingredients from essential oils, and identified 1,2,4-trimethoxybenzene (1,2,4-TTB) as a selective NLRP3 inflammasome inhibitor. We showed that 1,2,4-TTB (1 mM) markedly suppressed nigericin- or ATP-induced NLRP3 inflammasome activation, thus decreased caspase-1 activation and IL-1ß secretion in immortalized murine bone marrow-derived macrophages (iBMDMs) and in primary mouse microglia. Moreover, 1,2,4-TTB specifically inhibited the activation of NLRP3 inflammasome without affecting absent in melanoma 2 (AIM2) inflammasome activation. We further demonstrated that 1,2,4-TTB inhibited oligomerization of the apoptosis-associated speck-like protein containing a CARD (ASC) and protein-protein interaction between NLRP3 and ASC, thus blocking NLRP3 inflammasome assembly in iBMDMs and in primary mouse macrophages. In mice with experimental autoimmune encephalomyelitis (EAE), administration of 1,2,4-TTB (200 mg · kg-1 · d-1, i.g. for 17 days) significantly ameliorated EAE progression and demyelination. In conclusion, our results demonstrate that 1,2,4-TTB is an NLRP3 inflammasome inhibitor and attenuates the clinical symptom and inflammation of EAE, suggesting that 1,2,4-TTB is a potential candidate compound for treating NLRP3 inflammasome-driven diseases, such as multiple sclerosis.

Adipocytes promote breast tumorigenesis through TAZ-dependent secretion of Resistin.

Adipocytes have been implicated in breast tumor growth and stemness maintenance through secreted factors. However, the mechanisms by which these cytokines are regulated during diet-induced obesity and contribute to breast tumorigenesis remain largely unknown. Here we show that transcription cofactor TAZ in adipocytes is directly up-regulated by the free fatty acid/PPARγ axis upon dietary fat stimulation. TAZ knockdown alters the expression profile of a series of secreted proteins and attenuates the tumor-supporting function of adipocytes. Moreover, we identify Resistin, an adipose-derived hormone, as a functional downstream target of TAZ, which facilitates tumorigenesis, and its expression correlated with adipocyitc TAZ in triple-negative breast cancer samples. Further, Adiponectin-cre-mediated TAZ knockout in adipocytes mitigates breast tumor growth. Taken together, our findings highlight how diet-induced TAZ expression in adipocytes promotes tumorigenesis, suggesting promising cancer therapeutic targets.

Hematopoietic Cell Kinase (HCK) Is Essential for NLRP3 Inflammasome Activation and Lipopolysaccharide-Induced Inflammatory Response In Vivo.

Activation of the NLRP3 inflammasome results in caspase 1 cleavage, which subsequently leads to IL-1ß and IL-18 secretion, as well as pyroptosis, and aberrant activation of the inflammasome is involved in several diseases such as type 2 diabetes, atherosclerosis, multiple sclerosis, Parkinson's disease, and Alzheimer's disease. NLRP3 activity is regulated by various kinases. Genetic and pharmacological inhibition of the hematopoietic cell kinase (HCK), a member of the Src family of non-receptor tyrosine kinases (NRTKs) primarily expressed in myeloid cells, has previously been shown to ameliorate inflammation, indicating that it may be involved in the regulation of microglia function. However, the underlying mechanism is not known. Hence, in this study, we aimed to investigate the role of HCK in NLRP3 inflammasome activation. We demonstrated that HCK silencing inhibited NLRP3 inflammasome activation. Furthermore, the HCK-specific inhibitor, A419259, attenuated the release of IL-1ß and caspase 1(P20) from the macrophages and microglia and reduced the formation of the apoptosis-associated speck-like protein with a CARD domain (ASC) oligomer. We also observed that HCK binds to full length NLRP3 and its NBD(NACHT) and LRR domains, but not to the PYD domain. In vivo, the HCK inhibitor attenuated the LPS-induced inflammatory response in the liver of LPS-challenged mice. Collectively, these results suggested that HCK plays a critical role in NLRP3 inflammasome activation. Our results will enhance current understanding regarding the effectiveness of HCK inhibitors for treating acute inflammatory diseases.

Microglial autophagy defect causes parkinson disease-like symptoms by accelerating inflammasome activation in mice.

Microglial activation-induced neuroinflammation is closely associated with the development of Parkinson disease (PD). Macroautophagy/autophagy regulates many biological processes, but the role of autophagy in microglial activation during PD development remains largely unclear. In this study, we showed that deletion of microglial Atg5 caused PD-like symptoms in mice, characterized by impairment in motor coordination and cognitive learning, loss of tyrosine hydroxylase (TH) neurons, enhancement of neuroinflammation and reduction in dopamine levels in the striatum. Mechanistically, we found that inhibition of autophagy led to NLRP3 (NLR family pyrin domain containing 3) inflammasome activation via PDE10A (phosphodiesterase 10A)-cyclic adenosine monophosphate (cAMP) signaling in microglia, and the sequential upregulation of downstream IL1B/IL-1ß in turn increased the expression of MIF (macrophage migration inhibitory factor [glycosylation-inhibiting factor]), a pro-inflammatory cytokine. Inhibition of NLRP3 inflammasome activation by administration of MCC950, a specific inhibitor for NLRP3, decreased MIF expression and neuroinflammatory levels, and rescued the loss of TH neurons in the substantial nigra (SN). Interestingly, we found that serum MIF levels in PD patients were significantly elevated. Taken together, our results reveal an important role of autophagy in microglial activation-driven PD-like symptoms, thus providing potential targets for the clinical treatment of PD. Abbreviations: ATG: autophagy related; cAMP: cyclic adenosine monophosphate; cKO: conditional knockout; NOS2/INOS: nitric oxide synthase 2, inducible; IL1B: interleukin 1 beta; ITGAM/CD-11b: integrin alpha M/cluster of differentiation molecule 11B; MAP1LC3: microtubule-associated protein 1 light chain 3; MIF: macrophage migration inhibitory factor (glycosylation-inhibiting factor); NLRP3: NLR family pyrin domain containing 3; PBS: phosphate-buffered saline; PD: parkinson disease; PDE10A: phosphodiesterase 10A; SN: substantial nigra; TH: tyrosine hydroxylase; TNF: tumor necrosis factor; WT: wild type.

A novel m6A reader Prrc2a controls oligodendroglial specification and myelination.

While N6-methyladenosine (m6A), the most abundant internal modification in eukaryotic mRNA, is linked to cell differentiation and tissue development, the biological significance of m6A modification in mammalian glial development remains unknown. Here, we identify a novel m6A reader, Prrc2a (Proline rich coiled-coil 2 A), which controls oligodendrocyte specification and myelination. Nestin-Cre-mediated knockout of Prrc2a induces significant hypomyelination, decreased lifespan, as well as locomotive and cognitive defects in a mouse model. Further analyses reveal that Prrc2a is involved in oligodendrocyte progenitor cells (OPCs) proliferation and oligodendrocyte fate determination. Accordingly, oligodendroglial-lineage specific deletion of Prrc2a causes a similar phenotype of Nestin-Cre-mediated deletion. Combining transcriptome-wide RNA-seq, m6A-RIP-seq and Prrc2a RIP-seq analysis, we find that Olig2 is a critical downstream target gene of Prrc2a in oligodendrocyte development. Furthermore, Prrc2a stabilizes Olig2 mRNA through binding to a consensus GGACU motif in the Olig2 CDS (coding sequence) in an m6A-dependent manner. Interestingly, we also find that the m6A demethylase, Fto, erases the m6A modification of Olig2 mRNA and promotes its degradation. Together, our results indicate that Prrc2a plays an important role in oligodendrocyte specification through functioning as a novel m6A reader. These findings suggest a new avenue for the development of therapeutic strategies for hypomyelination-related neurological diseases.

The Function of the Mitochondrial Calcium Uniporter in Neurodegenerative Disorders.

The mitochondrial calcium uniporter (MCU)-a calcium uniporter on the inner membrane of mitochondria-controls the mitochondrial calcium uptake in normal and abnormal situations. Mitochondrial calcium is essential for the production of adenosine triphosphate (ATP); however, excessive calcium will induce mitochondrial dysfunction. Calcium homeostasis disruption and mitochondrial dysfunction is observed in many neurodegenerative disorders. However, the role and regulatory mechanism of the MCU in the development of these diseases are obscure. In this review, we summarize the role of the MCU in controlling oxidative stress-elevated mitochondrial calcium and its function in neurodegenerative disorders. Inhibition of the MCU signaling pathway might be a new target for the treatment of neurodegenerative disorders.

Autophagy regulates MAVS signaling activation in a phosphorylation-dependent manner in microglia.

Mitochondrial antiviral signaling (MAVS) protein has an important role in antiviral immunity and autoimmunity. However, the pathophysiological role of this signaling pathway, especially in the brain, remains elusive. Here we demonstrated that MAVS signaling existed and mediated poly(I:C)-induced inflammation in the brain. Along with the MAVS signaling activation, there was an induction of autophagic activation. Autophagy negatively regulated the activity of MAVS through direct binding of LC3 to the LIR motif Y(9)xxI(12) of MAVS. We also found that c-Abl kinase phosphorylated MAVS and regulated its interaction with LC3. Interestingly, tyrosine phosphorylation of MAVS was required for downstream signaling activation. Importantly, in vivo data showed that the deficiency of MAVS or c-Abl prevented MPTP-induced microglial activation and dopaminergic neuron loss. Together, our findings reveal the molecular mechanisms underlying the regulation of MAVS-dependent microglial activation in the nervous system, thus providing a potential target for the treatment of microglia-driven inflammatory brain diseases.

Amplified RLR signaling activation through an interferon-stimulated gene-endoplasmic reticulum stress-mitochondrial calcium uniporter protein loop.

Type I interferon (IFN-I) is critical for a host against viral and bacterial infections via induction of hundreds of interferon-stimulated genes (ISGs), but the mechanism underlying the regulation of IFN-I remains largely unknown. In this study, we first demonstrate that ISG expression is required for optimal IFN-ß levels, an effect that is further enhanced by endoplasmic reticulum (ER) stress. Furthermore, we identify mitochondrial calcium uniporter protein (MCU) as a mitochondrial antiviral signaling protein (MAVS)-interacting protein that is important for ER stress induction and amplified MAVS signaling activation. In addition, by performing an ectopic expression assay to screen a library of 117 human ISGs for effects on IFN-ß levels, we found that tumor necrosis factor receptor 1 (TNFR1) significantly increases IFN-ß levels independent of ER stress. Altogether, our findings suggest that MCU and TNFR1 are involved in the regulation of RIG-I-like receptors (RLR) signaling.

Thioredoxin 2 Is a Novel E2-Interacting Protein That Inhibits the Replication of Classical Swine Fever Virus.

UNLABELLED: The E2 protein of classical swine fever virus (CSFV) is an envelope glycoprotein that is involved in virus attachment and entry. To date, the E2-interacting cellular proteins and their involvement in viral replication have been poorly documented. In this study, thioredoxin 2 (Trx2) was identified to be a novel E2-interacting partner using yeast two-hybrid screening from a porcine macrophage cDNA library. Trx2 is a mitochondrion-associated protein that participates in diverse cellular events. The Trx2-E2 interaction was further confirmed by glutathione S-transferase (GST) pulldown, in situ proximity ligation, and laser confocal assays. The thioredoxin domain of Trx2 and the asparagine at position 37 (N37) in the E2 protein were shown to be critical for the interaction. Silencing of the Trx2 expression in PK-15 cells by small interfering RNAs significantly promotes CSFV replication, and conversely, overexpression of Trx2 markedly inhibits viral replication of the wild-type (wt) CSFV and to a greater extent that of the CSFV N37D mutant, which is defective in binding Trx2. The wt CSFV but not the CSFV N37D mutant was shown to reduce the Trx2 protein expression in PK-15 cells. Furthermore, we demonstrated that Trx2 increases nuclear factor kappa B (NF-κB) promoter activity by promoting the nuclear translocation of the p65 subunit of NF-κB. Notably, activation of the NF-κB signaling pathway induced by tumor necrosis factor alpha (TNF-α) significantly inhibits CSFV replication in PK-15 cells, whereas blocking the NF-κB activation in Trx2-overexpressing cells no longer suppresses CSFV replication. Taken together, our findings reveal that Trx2 inhibits CSFV replication via the NF-κB signaling pathway. IMPORTANCE: Thioredoxin 2 (Trx2) is a mitochondrion-associated protein that participates in diverse cellular events, such as antioxidative and antiapoptotic processes and the modulation of transcription factors. However, little is known about the involvement of Trx2 in viral replication. Here, we investigated, for the first time, the role of Trx2 in the replication of classical swine fever virus (CSFV), a devastating pestivirus of pigs. By knockdown and overexpression, we showed that Trx2 negatively regulates CSFV replication. Notably, we demonstrated that Trx2 inhibits CSFV replication by promoting the nuclear translocation of the p65 subunit of NF-κB, a key regulator of the host's innate immunity and inflammatory response. Our findings reveal a novel role of Trx2 in the host's antiviral response and provide new insights into the complex mechanisms by which CSFV interacts with the host cell.

Beta-actin interacts with the E2 protein and is involved in the early replication of classical swine fever virus.

The E2 glycoprotein of classical swine fever virus (CSFV) is involved in viral infection and induction of neutralizing antibodies. Little is known about how E2 interacts with host cells. To understand the cellular factors involved in viral replication cycle, E2 was used as bait in yeast two-hybrid screens, resulting in the identification of ß-actin as a potential E2-interacting partner. E2-ß-actin interaction was confirmed by co-immunoprecipitation, GST pulldown, laser confocal and bimolecular fluorescence complementation assays. The E2-interacting domain of ß-actin was mapped to amino acids (aa) 95-188 and two ß-actin-interacting regions were identified in E2 (aa 182-261 and aa 262-341). Knockdown of ß-actin by RNA interference and disruption of filamentous ß-actin with cytochalasin D at 4h post-infection caused a significant reduction of viral RNA copies and titers. Collectively, the results indicated that ß-actin is involved in the early replication of CSFV.

Comprehensive evaluation of the adenovirus/alphavirus-replicon chimeric vector-based vaccine rAdV-SFV-E2 against classical swine fever.

Classical swine fever (CSF) is an economically important, highly contagious swine disease caused by classical swine fever virus (CSFV). Marker vaccines and companion serological diagnostic tests are thought to be a promising strategy for future control and eradication of CSF. Previously, we have demonstrated that an adenovirus-vectored Semliki forest virus replicon construct expressing the E2 glycoprotein from CSFV, rAdV-SFV-E2, induced sterile immunity against a lethal CSFV challenge. In this study, we further evaluated the vaccine with respect to its safety, number and dose of immunization, and effects of maternally derived antibodies, re-immunization of the vaccine or co-administration with pseudorabies vaccine on the vaccine efficacy. The results showed that: (1) the vaccine was safe for mice, rabbits and pigs; (2) two immunizations with a dose as low as 6.25×10(5) TCID(50) or a single immunization with a dose of 10(7) TCID(50) rAdV-SFV-E2 provided complete protection against a lethal CSFV challenge; (3) maternally derived antibodies had no inhibitory effects on the efficacy of the vaccine; (4) the vaccine did not induce interfering anti-vector immunity; and (5) co-administration of rAdV-SFV-E2 with a live pseudorabies vaccine induced antibodies and protection indistinguishable from immunization with either vaccine administered alone. Taken together, the chimeric vaccine represents a promising marker vaccine candidate for control and eradication of CSF.