NIR Light-Activated and RGD-Conjugated Ultrasmall Fe/PPy Nanopolymers for Enhanced Tumor Photothermal Ferrotherapy and MR Imaging.

Iron-based nanomaterials have shown great promise for tumor ferrotherapy in recent years. However, nanoparticle-induced ferroptosis has low therapeutic efficacy owing to unsatisfactory Fenton reaction activity in a typical tumor microenvironment. In this study, NIR light-activated Fe/PPy-RGD nanopolymers were developed to combine photothermal therapy and ferrotherapy and achieve enhanced antitumor activity. Importantly, Fe/PPy-RGD exhibited excellent therapeutic performance under NIR light activation both in vitro and in vivo. Under irradiation with NIR light, the heat generated by Fe/PPy-RGD not only induced a therapeutic photothermal effect but also enhanced the release of iron ions and the Fenton reaction by inducing ferroptosis. Additionally, by virtue of RGD conjugation and its ultrasmall size, Fe/PPy-RGD could effectively accumulate at tumor sites in living mice after systemic administration and could be monitored via MR imaging. Hence, this study provides a promising approach for integrating ferrotherapy with photothermal therapy to achieve enhanced tumor treatment.

Hepatocyte-derived MANF mitigates ethanol-induced liver steatosis in mice via enhancing ASS1 activity and activating AMPK pathway.

Hepatic steatosis plays a detrimental role in the onset and progression of alcohol-associated liver disease (ALD). Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an evolutionarily conserved protein related to the unfolded protein response. Recent studies have demonstrated that MANF plays an important role in liver diseases. In this study, we investigated the role of MANF in ethanol-induced steatosis and the underlying mechanisms. We showed that the hepatic MANF expression was markedly upregulated in mouse model of ALD by chronic-plus-single-binge ethanol feeding. Moreover, after chronic-plus-binge ethanol feeding, hepatocyte-specific MANF knockout (HKO) mice displayed more severe hepatic steatosis and liver injury than wild-type (WT) control mice. Immunoprecipitation-coupled MS proteomic analysis revealed that arginosuccinate synthase 1 (ASS1), a rate-limiting enzyme in the urea cycle, resided in the same immunoprecipitated complex with MANF. Hepatocyte-specific MANF knockout led to decreased ASS1 activity, whereas overexpression of MANF contributed to enhanced ASS1 activity in vitro. In addition, HKO mice displayed unique urea cycle metabolite patterns in the liver with elevated ammonia accumulation after ethanol feeding. ASS1 is known to activate AMPK by generating an intracellular pool of AMP from the urea cycle. We also found that MANF supplementation significantly ameliorated ethanol-induced steatosis in vivo and in vitro by activating the AMPK signaling pathway, which was partly ASS1 dependent. This study demonstrates a new mechanism in which MANF acts as a key molecule in maintaining hepatic lipid homeostasis by enhancing ASS1 activity and uncovers an interesting link between lipid metabolism and the hepatic urea cycle under excessive alcohol exposure.

MANF ameliorates DSS-induced mouse colitis via restricting Ly6ChiCX3CR1int macrophage transformation and suppressing CHOP-BATF2 signaling pathway.

Mesencephalic astrocyte-derived neurotrophic factor (MANF), an endoplasmic reticulum stress-inducible secreting protein, has evolutionarily conserved immune-regulatory function that contributes to the negative regulation of inflammation in macrophages. In this study, we investigated the profiles of MANF in the macrophages of the patients with active inflammatory bowel disease (IBD) and the mice with experimental colitis, which was induced in both myeloid cell-specific MANF knockout mice and wild-type mice by 3% dextran sodium sulfate (DSS) for 7 days. We found that MANF expression was significantly increased in intestinal macrophages from both the mice with experimental colitis and patients with active IBD. DSS-induced colitis was exacerbated in myeloid cell-specific MANF knockout mice. Injection of recombinant human MANF (rhMANF, 10 mg·kg-1·d-1, i.v.) from D4 to D6 significantly ameliorated experimental colitis in DSS-treated mice. More importantly, MANF deficiency in myeloid cells resulted in a dramatic increase in the number of Ly6ChiCX3CRint proinflammatory macrophages in colon lamina propria of DSS-treated mice, and the proinflammatory cytokines and chemokines were upregulated as well. Meanwhile, we demonstrated that MANF attenuated Th17-mediated immunopathology by inhibiting BATF2-mediated innate immune response and downregulating CXCL9, CXCL10, CXCL11 and IL-12p40; MANF functioned as a negative regulator in inflammatory macrophages via inhibiting CHOP-BATF2 signaling pathway, thereby protecting against DSS-induced mouse colitis. These results suggest that MANF ameliorates colon injury by negatively regulating inflammatory macrophage transformation, which shed light on a potential therapeutic target for IBD.

Structural Basis of the Differential Function of the Two C. elegans Atg8 Homologs, LGG-1 and LGG-2, in Autophagy.

Multicellular organisms have multiple homologs of the yeast ATG8 gene, but the differential roles of these homologs in autophagy during development remain largely unknown. Here we investigated structure/function relationships in the two C. elegans Atg8 homologs, LGG-1 and LGG-2. lgg-1 is essential for degradation of protein aggregates, while lgg-2 has cargo-specific and developmental-stage-specific roles in aggregate degradation. Crystallography revealed that the N-terminal tails of LGG-1 and LGG-2 adopt the closed and open form, respectively. LGG-1 and LGG-2 interact differentially with autophagy substrates and Atg proteins, many of which carry a LIR motif. LGG-1 and LGG-2 have structurally distinct substrate binding pockets that prefer different residues in the interacting LIR motif, thus influencing binding specificity. Lipidated LGG-1 and LGG-2 possess distinct membrane tethering and fusion activities, which may result from the N-terminal differences. Our study reveals the differential function of two ATG8 homologs in autophagy during C. elegans development.

NADPH oxidase 4 contributes to TRPV4-mediated endothelium-dependent vasodilation in human arterioles by regulating protein phosphorylation of TRPV4 channels.

Impaired endothelium-dependent vasodilation has been suggested to be a key component of coronary microvascular dysfunction (CMD). A better understanding of endothelial pathways involved in vasodilation in human arterioles may provide new insight into the mechanisms of CMD. The goal of this study is to investigate the role of TRPV4, NOX4, and their interaction in human arterioles and examine the underlying mechanisms. Arterioles were freshly isolated from adipose and heart tissues obtained from 71 patients without coronary artery disease, and vascular reactivity was studied by videomicroscopy. In human adipose arterioles (HAA), ACh-induced dilation was significantly reduced by TRPV4 inhibitor HC067047 and by NOX 1/4 inhibitor GKT137831, but GKT137831 did not further affect the dilation in the presence of TRPV4 inhibitors. GKT137831 also inhibited TRPV4 agonist GSK1016790A-induced dilation in HAA and human coronary arterioles (HCA). NOX4 transcripts and proteins were detected in endothelial cells of HAA and HCA. Using fura-2 imaging, GKT137831 significantly reduced GSK1016790A-induced Ca2+ influx in the primary culture of endothelial cells and TRPV4-WT-overexpressing human coronary artery endothelial cells (HCAEC). However, GKT137831 did not affect TRPV4-mediated Ca2+ influx in non-phosphorylatable TRPV4-S823A/S824A-overexpressing HCAEC. In addition, treatment of HCAEC with GKT137831 decreased the phosphorylation level of Ser824 in TRPV4. Finally, proximity ligation assay (PLA) revealed co-localization of NOX4 and TRPV4 proteins. In conclusion, both TRPV4 and NOX4 contribute to ACh-induced dilation in human arterioles from patients without coronary artery disease. NOX4 increases TRPV4 phosphorylation in endothelial cells, which in turn enhances TRPV4-mediated Ca2+ entry and subsequent endothelium-dependent dilation in human arterioles.

Prp5-Spt8/Spt3 interaction mediates a reciprocal coupling between splicing and transcription.

Transcription and pre-mRNA splicing are coupled to promote gene expression and regulation. However, mechanisms by which transcription and splicing influence each other are still under investigation. The ATPase Prp5p is required for pre-spliceosome assembly and splicing proofreading at the branch-point region. From an open UV mutagenesis screen for genetic suppressors of prp5 defects and subsequent targeted testing, we identify components of the TBP-binding module of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex, Spt8p and Spt3p. Spt8Δ and spt3Δ rescue the cold-sensitivity of prp5-GAR allele, and prp5 mutants restore growth of spt8Δ and spt3Δ strains on 6-azauracil. By chromatin immunoprecipitation (ChIP), we find that prp5 alleles decrease recruitment of RNA polymerase II (Pol II) to an intron-containing gene, which is rescued by spt8Δ. Further ChIP-seq reveals that global effects on Pol II-binding are mutually rescued by prp5-GAR and spt8Δ. Inhibited splicing caused by prp5-GAR is also restored by spt8Δ. In vitro assays indicate that Prp5p directly interacts with Spt8p, but not Spt3p. We demonstrate that Prp5p's splicing proofreading is modulated by Spt8p and Spt3p. Therefore, this study reveals that interactions between the TBP-binding module of SAGA and the spliceosomal ATPase Prp5p mediate a balance between transcription initiation/elongation and pre-spliceosome assembly.

The momentous role of N6-methyladenosine in lung cancer.

Lung cancer is the leading cause of cancer death worldwide. Although diagnostic methods and targeted drugs have been rapidly developed in recent years, the underlying molecular mechanisms in the pathogenesis of lung cancer remain enigmatic. The N6-methyladenosine (m6 A) modification is the most common modification of messenger RNA in eukaryotes and plays critical roles in many diseases, especially cancers. Ectopic m6 A modification is associated with human carcinogenesis, including lung cancer. The m6 A modification is mediated by methyltransferases (writers) and demethylases (erasers) and indirectly affects biological processes through the recruitment of specific reader proteins (readers). Many studies have shown that m6 A writers, erasers, and readers serve as specific and sensitive biomarkers for lung cancer diagnosis, prognosis, and therapy. This review summarizes recent studies on the biological functions of the m6 A modification in lung cancer and discusses the potential application of m6 A regulators in lung cancer diagnosis and therapeutics.

Mesencephalic Astrocyte-Derived Neurotrophic Factor Inhibits Liver Cancer Through Small Ubiquitin-Related Modifier (SUMO)ylation-Related Suppression of NF-κB/Snail Signaling Pathway and Epithelial-Mesenchymal Transition.

BACKGROUND AND AIMS: Endoplasmic reticulum (ER) stress is associated with liver inflammation and hepatocellular carcinoma (HCC). However, how ER stress links inflammation and HCC remains obscure. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an ER stress-inducible secretion protein that inhibits inflammation by interacting with the key subunit of nuclear factor kappa light chain enhancer of activated B cells (NF-κB) p65. We hypothesized that MANF may play a key role in linking ER stress and inflammation in HCC. APPROACH AND RESULTS: Here, we found that MANF mRNA and protein levels were lower in HCC tissues versus adjacent noncancer tissues. Patients with high levels of MANF had better relapse-free survival and overall survival rates than those with low levels. MANF levels were also associated with the status of liver cirrhosis, advanced tumor-node-metastasis (TNM) stage, and tumor size. In vitro experiments revealed that MANF suppressed the migration and invasion of hepatoma cells. Hepatocyte-specific deletion of MANF accelerated N-nitrosodiethylamine (DEN)-induced HCC by up-regulating Snail1+2 levels and promoting epithelial-mesenchymal transition (EMT). MANF appeared in the nuclei and was colocalized with p65 in HCC tissues and in tumor necrosis factor alpha (TNF-α)-treated hepatoma cells. The interaction of p65 and MANF was also confirmed by coimmunoprecipitation experiments. Consistently, knockdown of MANF up-regulated NF-κB downstream target genes TNF-α, interleukin (IL)-6 and IL-1α expression in vitro and in vivo. Finally, small ubiquitin-related modifier 1 (SUMO1) promoted MANF nuclear translocation and enhanced the interaction of MANF and p65. Mutation of p65 motifs for SUMOylation abolished the interaction of p65 and MANF. CONCLUSIONS: MANF plays an important role in linking ER stress and liver inflammation by inhibiting the NF-κB/Snail signal pathway in EMT and HCC progression. Therefore, MANF may be a cancer suppressor and a potential therapeutic target for HCC.

Hepatocyte-derived MANF alleviates hepatic ischaemia-reperfusion injury via regulating endoplasmic reticulum stress-induced apoptosis in mice.

BACKGROUND: Endoplasmic reticulum (ER) perturbations are novel subcellular effectors involved in the ischaemia-reperfusion injury. As an ER stress-inducible protein, mesencephalic astrocyte-derived neurotrophic factor (MANF) has been proven to be increased during ischaemic brain injury. However, the role of MANF in liver ischaemia reperfusion (I/R) injury has not yet been studied. METHODS: To investigate the role of MANF in the process of liver ischaemia-reperfusion, Hepatocyte-specific MANF knockout (MANFhep-/- ) mice and their wild-type (WT) littermates were used in our research. Mice partial (70%) warm hepatic I/R model was established by vascular occlusion. We detected the serum levels of MANF in both liver transplant patients and WT mice before and after liver I/R injury. Recombinant human MANF (rhMANF) was injected into the tail vein before 1 hour occlusion. AST, ALT and Suzuki score were used to evaluate the extent of I/R injury. OGD/R test was performed on primary hepatocytes to simulate IRI in vitro. RNA sequence and RT-PCR were used to detect the cellular signal pathway activation while MANF knockout. RESULTS: We found that MANF expression and secretion are dramatically up-regulated during hepatic I/R. Hepatocyte-specific MANF knockout aggravates the I/R injury through the over-activated ER stress. The systemic administration of rhMANF before ischaemia has the potential to ameliorate I/R-triggered UPR and liver injury. Further study showed that MANF deficiency activated ATF4/CHOP and JNK/c-JUN/CHOP pathways, and rhMANF inhibited the activation of the two proapoptotic pathways caused by MANF deletion. CONCLUSION: Collectively, our study unravels a previously unknown relationship among MANF, UPR and hepatic I/R injury.

Xanthatin induces glioma cell apoptosis and inhibits tumor growth via activating endoplasmic reticulum stress-dependent CHOP pathway.

Xanthatin is a natural sesquiterpene lactone purified from Xanthium strumarium L., which has shown prominent antitumor activity against a variety of cancer cells. In the current study, we investigated the effect of xanthatin on the growth of glioma cells in vitro and in vivo, and elucidated the underlying mechanisms. In both rat glioma C6 and human glioma U251 cell lines, xanthatin (1-15 µM) dose-dependently inhibited cell viability without apparent effect on the cell cycle. Furthermore, xanthatin treatment dose-dependently induced glioma cell apoptosis. In nude mice bearing C6 glioma tumor xenografts, administration of xanthatin (10, 20, 40 mg·kg-1·d-1, ip, for 2 weeks) dose-dependently inhibited the tumor growth, but did not affect the body weight. More importantly, xanthatin treatment markedly increased the expression levels of the endoplasmic reticulum (ER) stress-related markers in both the glioma cell lines as well as in C6 xenografts, including glucose-regulated protein 78, C/EBP-homologous protein (CHOP), activating factor 4, activating transcription factor 6, spliced X-box binding protein-1, phosphorylated protein kinase R-like endoplasmic reticulum kinase, and phosphorylated eukaryotic initiation factor 2a. Pretreatment of C6 glioma cells with the ER stress inhibitor 4-phenylbutyric acid (4-PBA, 7 mM) or knockdown of CHOP using small interfering RNA significantly attenuated xanthatin-induced cell apoptosis and increase of proapoptotic caspase-3. These results demonstrate that xanthatin induces glioma cell apoptosis and inhibits tumor growth via activating the ER stress-related unfolded protein response pathway involving CHOP induction. Xanthatin may serve as a promising agent in the treatment of human glioma.

Microglial proliferation and monocyte infiltration contribute to microgliosis following status epilepticus.

Microglial activation has been recognized as a major contributor to inflammation of the epileptic brain. Seizures are commonly accompanied by remarkable microgliosis and loss of neurons. In this study, we utilize the CX3CR1GFP/+ CCR2RFP/+ genetic mouse model, in which CX3CR1+ resident microglia and CCR2+ monocytes are labeled with GFP and RFP, respectively. Using a combination of time-lapse two-photon imaging and whole-cell patch clamp recording, we determined the distinct morphological, dynamic, and electrophysiological characteristics of infiltrated monocytes and resident microglia, and the evolution of their behavior at different time points following kainic acid-induced seizures. Seizure activated microglia presented enlarged somas with less ramified processes, whereas, infiltrated monocytes were smaller, highly motile cells that lacked processes. Moreover, resident microglia, but not infiltrated monocytes, proliferate locally in the hippocampus after seizure. Microglial proliferation was dependent on the colony-stimulating factor 1 receptor (CSF-1R) pathway. Pharmacological inhibition of CSF-1R reduced seizure-induced microglial proliferation, which correlated with attenuation of neuronal death without altering acute seizure behaviors. Taken together, we demonstrated that proliferation of activated resident microglia contributes to neuronal death in the hippocampus via CSF-1R after status epilepticus, providing potential therapeutic targets for neuroprotection in epilepsy.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) protects against Aß toxicity via attenuating Aß-induced endoplasmic reticulum stress.

BACKGROUND: Extracellular accumulation of amyloid ß-peptide (Aß) is one of pathological hallmarks of Alzheimer's disease (AD) and contributes to the neuronal loss. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) stress-inducible neurotrophic factor. Many groups, including ours, have proved that MANF rescues neuronal loss in several neurological disorders, such as Parkinson's disease and cerebral ischemia. However, whether MANF exerts its protective effect against Aß neurotoxicity in AD remains unknown. METHODS: In the present study, the characteristic expressions of MANF in Aß1-42-treated neuronal cells as well as in the brains of APP/PS1 transgenic mice were analyzed by immunofluorescence staining, qPCR, and Western blot. The effects of MANF overexpression, MANF knockdown, or recombination human MANF protein (rhMANF) on neuron viability, apoptosis, and the expression of ER stress-related proteins following Aß1-42 exposure were also investigated. RESULTS: The results showed the increased expressions of MANF, as well as ER stress markers immunoglobulin-binding protein (BiP) and C/EBP homologous protein (CHOP), in the brains of the APP/PS1 transgenic mice and Aß1-42-treated neuronal cells. MANF overexpression or rhMANF treatment partially protected against Aß1-42-induced neuronal cell death, associated with marked decrease of cleaved caspase-3, whereas MANF knockdown with siRNA aggravated Aß1-42 cytotoxicity including caspase-3 activation. Further study demonstrated that the expressions of BiP, ATF6, phosphorylated-IRE1, XBP1s, phosphorylated-eIF2α, ATF4, and CHOP were significantly downregulated by MANF overexpression or rhMANF treatment in neuronal cells following Aß1-42 exposure, whereas knockdown of MANF has the opposite effect. CONCLUSIONS: These findings demonstrate that MANF may exert neuroprotective effects against Aß-induced neurotoxicity through attenuating ER stress, suggesting that an applicability of MANF as a therapeutic candidate for AD.

Dexmedetomidine Promotes SH-SY5Y Cell Resistance Against Impairment of Iron Overload by Inhibiting NF-κB Pathways.

Iron overload is a common pathophysiological state underlying many diseases that has a detrimental influence on cells. The protective effects of Dexmedetomidine (Dex), a high selective alpha-2-adrenoceptor agonist, have been revealed through many experimental models, whereas no study reports its effects on an iron overload model. To elucidate these effects, we used FeCl2 with or without Dex to treat SH-SY5Y cells for 24 h and then detected indicators of oxidative stress, inflammation and apoptosis and investigated possible mechanisms further. After treatment with FeCl2 for 24 h, cell viability decreased in a dose dependent manner, and Dex promoted cell survival in FeCl2 incubation, also in a dose-dependent manner. Compared with the FeCl2 group, 20 µM Dex significantly attenuated ROS accumulation, reduced pro-inflammatory cytokine expression, and inhibited apoptosis. Furthermore, 20 µM concentration of Dex remarkably downregulated the expression of pro-apoptotic protein and activation of caspase 3 while increasing anti-apoptotic protein expression. Additionally, Dex also effectively suppressed the expression of NF-κB and its activation. In conclusion, Dex exerted anti-oxidative stress, anti-inflammation, and anti-apoptosis effects on FeCl2-treated SH-SY5Y cells, possibly by inhibiting NF-κB signaling pathway.

The antitumour growth and antiangiogenesis effects of xanthatin in murine glioma dynamically evaluated by dynamic contrast-enhanced magnetic resonance imaging.

To investigate the suppressive effects of xanthatin on glioma growth in a nude mouse xenograft model and rat orthotopic implantation model using magnetic resonance imaging (MRI) to dynamically monitor the antitumour growth and antiangiogenesis effects of xanthatin. The nude mouse xenograft tumour model and rat orthotopic implantation model were established to observe the antitumour effects of xanthatin in vivo. In the rat orthotopic implanted tumour model, MRI scanning was used to dynamically monitor the antitumour growth effect and evaluate the antiangiogenesis effect of xanthatin. We found that xanthatin at a dose of 0.4 mg/10 g dramatically decreased the growth of xenograft tumours in nude mice. The antiangiogenesis effect of xanthatin C6 glioma was evaluated by dynamic contrast-enhanced (DCE) MRI via comparison of the volume transfer constant (Ktrans ) value, a parameter that reflects vessel permeability. We found that xanthatin at the doses of 8 and 16 mg/kg significantly decreased the Ktrans value, which suggests that xanthatin has antiangiogenesis effects. These data demonstrate the suppressive effects of xanthatin on C6 glioma occur via antiangiogenesis. Meanwhile, this study also provides evidence for the application of quantitative parameters of DCE-MRI for dynamically evaluating the growth and angiogenesis of intracranial tumours and for experimental and clinical research.

Roles of SAMHD1 in antiviral defense, autoimmunity and cancer.

The enzyme, sterile α motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1) diminishes infection of human immunodeficiency virus type 1 (HIV-1) by hydrolyzing intracellular deoxynucleotide triphosphates (dNTPs) in myeloid cells and resting CD4+ T cells. This dNTP degradation reduces the dNTP concentration to a level insufficient for viral cDNA synthesis, thereby inhibiting retroviral replication. This antiviral enzymatic activity can be inhibited by viral protein X (Vpx). The HIV-2/SIV Vpx causes degradation of SAMHD1, thus interfering with the SAMHD1-mediated restriction of retroviral replication. Recently, SAMHD1 has been suggested to restrict HIV-1 infection by directly digesting genomic HIV-1 RNA through a still controversial RNase activity. Here, we summarize the current knowledge about structure, antiviral mechanisms, intracellular localization, interferon-regulated expression of SAMHD1. We also describe SAMHD1-deficient animal models and an antiviral drug on the basis of disrupting proteasomal degradation of SAMHD1. In addition, the possible roles of SAMHD1 in regulating innate immune sensing, Aicardi-Goutières syndrome and cancer are discussed in this review.

DPF2 regulates OCT4 protein level and nuclear distribution.

The amount of transcription factor OCT4 is strictly regulated. A tight regulation of OCT4 levels is crucial for mammalian embryonic development and oncogenesis. However, the mechanisms underlying regulation of OCT4 protein expression and nuclear distribution are largely unknown. Here, we report that DPF2, a plant homeodomain (PHD) finger protein, is upregulated during H9 cell differentiation induced by retinoic acid. Endogenous interaction between DPF2 and OCT4 in P19 cells was revealed by an immunoprecipitation assay. GST-pull down assay proved that OCT4 protein in H9 cells and recombinant OCT4 can precipitate with DPF2 in vitro. In vitro ubiquitination assay demonstrated DPF2 might serve as an E3 ligase. Knock down of dpf2 using siRNA increased OCT4 protein level and stability in P19 cells. DPF2 siRNAs also up-regulates OCT4 but not NANOG in H9 cells. However, RA fails to downregulates OCT4 protein level in cells infected by lenitviruses containing DPF2 siRNA. Moreover, overexpression of both DPF2 and OCT4 in 293 cells proved the DPF2-OCT4 interaction. DPF2 but not PHD2 mutant DPF2 enhanced ubiquitination and degradation of OCT4 in 293 cells co-expressed DPF2 and OCT4. Both wild type DPF2 and PHD2 mutant DPF2 redistributes nuclear OCT4 without affecting DPF2-OCT4 interaction. Further analysis indicated that DPF2 decreases monomeric and mono-ubiquitinated OCT4, assembles poly-ubiquitin chains on OCT4 mainly through Ub-K48 linkage. These findings contribute to an understanding of how OCT4 protein level and nuclear distribution is regulated by its associated protein.

MRI Dynamically Evaluates the Therapeutic Effect of Recombinant Human MANF on Ischemia/Reperfusion Injury in Rats.

As an endoplasmic reticulum (ER) stress-inducible protein, mesencephalic astrocyte-derived neurotrophic factor (MANF) has been proven to protect dopaminergic neurons and nondopaminergic cells. Our previous studies had shown that MANF protected against ischemia/reperfusion injury. Here, we developed a magnetic resonance imaging (MRI) technology to dynamically evaluate the therapeutic effects of MANF on ischemia/reperfusion injury. We established a rat focal ischemic model by using middle cerebral artery occlusion (MCAO). MRI was performed to investigate the dynamics of lesion formation. MANF protein was injected into the right lateral ventricle at 3 h after reperfusion following MCAO for 90 min, when the obvious lesion firstly appeared according to MRI investigation. T2-weighted imaging for evaluating the therapeutic effects of MANF protein was performed in ischemia/reperfusion injury rats on Days 1, 2, 3, 5, and 7 post-reperfusion combined with histology methods. The results indicated that the administration of MANF protein at the early stage after ischemia/reperfusion injury decreased the mortality, improved the neurological function, reduced the cerebral infarct volume, and alleviated the brain tissue injury. The findings collected from MRI are consistent with the morphological and pathological changes, which suggest that MRI is a useful technology for evaluating the therapeutic effects of drugs.

Toxoplasma gondii virulence factor ROP18 inhibits the host NF-κB pathway by promoting p65 degradation.

The obligate intracellular parasite Toxoplasma gondii secretes effector molecules into the host cell to modulate host immunity. Previous studies have shown that T. gondii could interfere with host NF-κB signaling to promote their survival, but the effectors of type I strains remain unclear. The polymorphic rhoptry protein ROP18 is a key serine/threonine kinase that phosphorylates host proteins to modulate acute virulence. Our data demonstrated that the N-terminal portion of ROP18 is associated with the dimerization domain of p65. ROP18 phosphorylates p65 at Ser-468 and targets this protein to the ubiquitin-dependent degradation pathway. The kinase activity of ROP18 is required for p65 degradation and suppresses NF-κB activation. Consistently, compared with wild-type ROP18 strain, ROP18 kinase-deficient type I parasites displayed a severe inability to inhibit NF-κB, culminating in the enhanced production of IL-6, IL-12, and TNF-α in infected macrophages. In addition, studies have shown that transgenic parasites carrying kinase-deficient ROP18 induce M1-biased activation. These results demonstrate for the first time that the virulence factor ROP18 in T. gondii type I strains is responsible for inhibiting the host NF-κB pathway and for suppressing proinflammatory cytokine expression, thus providing a survival advantage to the infectious agent.

Small ubiquitin-related modifier 2/3 interacts with p65 and stabilizes it in the cytoplasm in HBV-associated hepatocellular carcinoma.

BACKGROUND: SUMOylation, an important post-translational modification, associates with the development of hepatocellular carcinoma (HCC). p65, one of the most important subunits of NF-κB, is a key regulator in the development of HCC and has been reported to be SUMOylated by exogenous small ubiquitin-related modifier 3 (SUMO3) in HEK 293T cells. However, the relationship between p65 and SUMO2/3 in HCC remains unknown. This study was to investigate the interaction between p65 and SUMO2/3 and explore the potential roles involved in HCC. METHODS: The expressions of p65 and SUMO2/3 in the liver tissues were detected by using immunohistochemistry. We performed double-labeled immunofluorescence and co-immunoprecipitation assay to verify the interaction between p65 and SUMO2/3. The extraction of nuclear and cytoplasmic proteins was performed, and the subcellular localization of p65 was detected. The proliferation and migration of hepatoma cells were observed using MTT, colony formation, and transwell assays. RESULTS: We found a strong SUMO2/3-positive immunoreactivity in the cytoplasm in the non-tumor tissues of HCC. However, SUMO2/3 level was down regulated in the tumor tissues as compared with the adjacent non-tumor tissues. In accordance with this finding, p65 was up regulated in the adjacent non-tumor tissues and almost localized in the cytoplasm. There was a close correlation between SUMO2/3 and p65 expressions in the liver tissues (R = 0.800, p = 0.006). The interaction between p65 and SUMO2/3 was verified by co-immunoprecipitation and double-labeled immunofluorescent assays. TNF-α (10 ng/ml) treatment for 30 min not only up regulated the cytoplasmic conjugated SUMO2/3, but also enhanced SUMO2/3-p65 interaction. Furthermore, we found that SUMO2/3 up regulated the cytoplasmic p65 protein level in a dose-dependent manner, but not affected its mRNA level. The increase of p65 protein by SUMO2/3 was abolished by MG132 treatment, a reversible inhibitor of proteasome. Meanwhile, TNF-α-induced increase of SUMO2/3-conjugated p65 was along with the reduction of the ubiquitin-conjugated p65. The further study showed that SUMO2/3 over-expression decreased the proliferative ability of hepatoma cells, but did not affect the migration. CONCLUSION: SUMO2/3-p65 interaction may be a novel mechanism involved in the transformation from chronic hepatitis B to HCC via stabilizing cytoplasmic p65, which might shed light on understanding the tumorigenesis and development.