Tumor therapeutics work as stress inducers to enhance tumor sensitivity to NK cell cytolysis by upregulating NKp30 ligand B7-H6 [Cell Biology]

October 15th, 2015 by Cao, G., Wang, J., Zheng, X., Wei, H., Tian, Z., Sun, R.

Immune cells are believed to participate in initiating anti-tumor effects during regular tumor therapy such as chemotherapy, radiation, hyperthermia and cytokine injection. One of the mechanisms underlying this process is the expression of so-called stress-inducible immunostimulating ligands. Although the activating receptor NKG2D has been proven to play roles in tumor therapy through targeting its ligands, the role of NKp30, another key activating receptor, is seldom addressed. In this study, we found that the NKp30 ligand B7-H6 was widely expressed in tumor cells and closely correlated to their susceptibility to NK cell lysis. Further studies showed that treatment of tumor cells with almost all standard tumor therapeutics, including chemotherapy (cisplatin, 5-fluorouracil), radiation therapy, non-lethal heat shock, and cytokine therapy (TNF-α), could upregulate the expression of B7-H6 in tumor cells and enhance tumor sensitivity to NK cell cytolysis. B7-H6 shRNA treatment effectively dampened sensitization of tumor cells to NK-mediated lysis. Our study not only reveals the possibility that tumor therapeutics work as stress inducers to enhance tumor sensitivity to NK cell cytolysis but also suggests that B7-H6 could be a potential target for tumor therapy in the future.
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ABC Transport System Solute Binding Protein-Guided Identification of Novel D-Altritol and Galactitol Catabolic Pathways in Agrobacterium tumefaciencs C58 [Metabolism]

October 15th, 2015 by

Innovations in the discovery of the functions of uncharacterized proteins/enzymes have become increasingly important as advances in sequencing technology flood protein databases with an exponentially growing number of open reading frames. This study documents one such innovation developed by the Enzyme Function Initiative (EFI; U54GM093342), the use of solute binding proteins (SBPs) for transport systems to identify novel metabolic pathways. In a previous study, this strategy was applied to the tripartite ATP-independent periplasmic (TRAP) transporters. Here, we apply this strategy to the ATP-binding cassette (ABC) transporters, and report the discovery of novel catabolic pathways for D-altritol and galactitol in Agrobacterium tumefaciens C58. These efforts resulted in the description of three novel enzymatic reactions: 1) oxidation of D-altritol to D-tagatose via a dehydrogenase in Pfam family PF00107, a previously unknown reaction; 2) phosphorylation of D-tagatose to D-tagatose 6-phosphate via a kinase in Pfam family PF00294, a previously orphan EC number; and 3) epimerization of D-tagatose 6-phosphate C-4 to D-fructose 6-phosphate via a member of Pfam family PF08013, another previously unknown reaction. The epimerization reaction catalyzed by a member of PF08013 is especially noteworthy, because the functions of members of PF08013 have been unknown. These discoveries were assisted by two synergistic bioinformatics web tools made available by the Enzyme Function Initiative: the EFI-Enzyme Similarity Tool (EFI-EST) and the EFI-Genome Neighborhood Tool (EFI-GNT).
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The Nogo/ Nogo-66 receptor (NgR) signal is involved in neuroinflammation through the regulation of microglia inflammatory mediator expression [Immunology]

October 15th, 2015 by

Microglia has been proposed to play a pivotal role in the inflammation response of the central nervous system (CNS) by expressing a range of proinflammatory enzymes and cytokines under pathological stimulus. Our previous study has confirmed that Nogo receptor (NgR), an axon outgrowth inhibition receptor, is also expressed on microglia and regulate cell adhesion and migration behavior in vitro. In the present study, we further investigated the pro-inflammatory effects and possible mechanisms of Nogo on microglia in vitro. In this study, Nogo peptide, Nogo-P4, a 25-aa core inhibitory peptide sequence of Nogo-66, was used. We found that Nogo-P4 was able to induce the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and the release of proinflammatory cytokines including interleukin-1β (IL-1β), Tumor necrosis factor-α (TNF-α), nitric oxide (NO) and Prostaglandin E2 (PGE2) in microglia, which could be reversed by Nogo-66(1-40) antagonist peptide (NEP1-40), phosphatidylinositol-specific phospholipase C (PI-PLC) or NgR siRNA treatment. After Nogo-P4 stimulated microglia the phosphorylation levels of NF-κB and STAT3 were increased obviously, which further mediated microglia expressing proinflammatory factors induced by Nogo-P4. Taken together, we concluded that Nogo peptide could directly take part in CNS inflammatory process by influencing the expression of proinflammatory factors in microglia, which was related to the NF-κB and STAT3 signal pathways. Besides neurite outgrowth restriction, the Nogo/NgR signal might be involved in multiple processes in various inflammation-associated CNS diseases.
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Glycogen Synthase Kinase-3{beta}-mediated Phosphorylation in the Most C-terminal Region of Protein Interacting with C Kinase 1 (PICK1) Regulates the Binding of PICK1 to Glutamate Receptor Subunit GluA2 [Molecular Bases of Disease]

October 15th, 2015 by

Protein interacting with C kinase 1 (PICK1) is a synaptic protein interacting with the AMPA receptor subunits GluA2/3. The interaction between GluA2 and PICK1 is required for the removal of GluA2 from the synaptic plasma membrane during long-term depression (LTD). It has been suggested that glycogen synthase kinase-3β(GSK-3β) is activated during LTD, but the relationships between GluA2, PICK1, and GSK-3β are not well understood. In particular, the substrate(s) of GSK-3β have not been determined yet. Here, we showed that PICK1 was a substrate of GSK-3β. We found that Ser339, Ser342, Ser412, and Ser416 of PICK1 were putative GSK-3β-mediated phosphorylation sites. Among these sites, Ser416 played a crucial role in regulating the interaction between GluA2 and PICK1. We showed that replacing Ser416 with Ala disrupted the GluA2-PICK1 interaction, while substituting Ser416 with Glu or Asp retained this interaction. However, the deletion of Ser416 did not affect the GluA2-PICK1 interaction, and the substitution of Ser416 with Ala did not alter the PICK1-PICK1 interaction. Using image analysis in COS-7 cells with AcGFP1-fused PICK1, we showed that substitution of Ser416 with Ala increased the formation of AcGFP1-positive clusters, suggesting an increase in the association of PICK1 with the membrane. This may have resulted in the dissociation of the GluA2-PICK1 complexes. Our results indicated that GSK-3β-mediated phosphorylation of PICK1 at Ser416 was required for its association with the AMPA receptor subunit. Thus, the GSK-3β-mediated phosphorylation of PICK1 may be a regulating factor during LTD induction.
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Human MageB2 Expression Enhances E2F Activity, Cell Proliferation And Resistance To Ribotoxic Stress [Cell Biology]

October 14th, 2015 by

MageB2 belongs to the MAGE-I (Melanoma Antigen GEne) family of tumor-specific antigens. Expression of this gene has been detected in human tumors of different origins. However, little is known about the protein function and how its expression impacts on tumor cell phenotypes. In this work we found that human MageB2 protein promotes tumor cell proliferation in a p53-independent fashion as observed both in cultured cells and growing tumors in mice. Gene expression analysis showed that MageB2 enhances the activity of E2Fs transcription factors. Mechanistically, the activation of E2Fs is related to the ability of MageB2 to interact with the E2F inhibitor, HDAC1. Cellular distribution of MageB2 protein includes the nucleoli; nevertheless ribotoxic drugs rapidly promote its nucleolar exit. We showed that MageB2 counteracts the E2F inhibition by ribosomal proteins independently of Mdm2 expression. Importantly, MageB2 plays a critical role in impairing cell cycle arrest in response to Actinomycin D. Data presented here support a relevant function for human MageB2 in cancer cells both in cycling and stressed conditions, thus presenting a distinct functional feature with respect to other characterized MAGE proteins.

Slow off-rates and strong product binding are required for processivity and efficient degradation of recalcitrant chitin by family 18 chitinases [Enzymology]

October 14th, 2015 by Kurašin, M., Kuusk, S., Kuusk, P., Sorlie, M., Valȷamae, P.

Processive glycoside hydrolases are the key components of enzymatic machineries that decompose recalcitrant polysaccharides, such as chitin and cellulose. The intrinsic processivity (PIntr) of cellulases has been shown to be governed by the rate constant of dissociation from polymer chain (koff). However, the reported koff values of cellulases are strongly dependent on the method used for their measurement. Here we developed a new method for determining koff, based on measuring the exchange rate of the enzyme between a non-labeled and a 14C-labeled polymeric substrate. The method was applied to the study of the processive chitinase ChiA from Serratia marcescens. In parallel, ChiA variants with weaker binding of N-acetyl-glucosamine unit either in the substrate binding site −3 (ChiA-W167A) or the product binding site +1 (ChiA-W275A) were studied. Both ChiA variants showed increased off-rates and lower apparent processivity on α-chitin. The rate of the production of insoluble reducing groups on the reduced α-chitin was an order of magnitude higher than koff, suggesting that the enzyme can initiate several processive runs without leaving the substrate. On crystalline chitin, the general activity of the wild type enzyme was higher and the difference was magnifying with hydrolysis time. On amorphous chitin, the variants clearly outperformed the wild type. A model is proposed, whereby strong interactions with polymer in the substrate binding sites (low off-rates) and strong binding of the product in the product binding sites (high pushing potential) are required for the removal of obstacles, like disintegration of chitin microfibrils.
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Glucosamine modulates T cell differentiation through downregulating N-linked glycosylation of CD25 [Glycobiology and Extracellular Matrices]

October 14th, 2015 by

Glucosamine has immunomodulatory effects on autoimmune diseases. However, the mechanism(s) through which glucosamine modulates different T cell subsets and diseases remain unclear. We demonstrate that glucosamine impedes Th1, Th2, and iTreg but promotes Th17 differentiation through downregulating N-linked glycosylation of CD25 and subsequently inhibiting its downstream Stat5 signaling in a dose-dependent manner. The effect of glucosamine on T helper cell differentiation was similar to that induced by anti-IL-2 treatment, further supporting an IL-2 signaling-dependent modulation. Interestingly, excess glucose rescued this glucosamine-mediated regulation, suggesting a functional competition between glucose and glucosamine. High-dose glucosamine significantly decreased Glut1 N-glycosylation in Th1-polarized cells. This finding suggests that both downregulated IL-2 signaling and Glut1-dependent glycolytic metabolism contribute to the inhibition of Th1 differentiation by glucosamine. Finally, glucosamine treatment inhibited Th1 cells in vivo, prolonged the survival of islet grafts in diabetic recipients, and exacerbated the severity of EAE. Taken together, our results indicate that glucosamine interferes with N-glycosylation of CD25, and thereby attenuates IL-2 downstream signaling. These effects suggest that glucosamine may be an important modulator of T cell differentiation and immune homeostasis.

The inflammasome adaptor ASC induces procaspase-8 death effector domain filaments [Signal Transduction]

October 14th, 2015 by

Inflammasomes mediate inflammatory and cell death responses to pathogens and cellular stress signals via activation of procaspases-1 and -8. During inflammasome assembly, activated receptors of the NLR or PYHIN family recruit the adaptor protein ASC and initiate polymerisation of its pyrin domain (PYD) into filaments. We show that ASC filaments in turn nucleate procaspase-8 death effector domain (DED) filaments in vitro and in vivo. Interaction between ASC PYD and procaspase-8 tandem DEDs optimally required both DEDs, and represents an unusual heterotypic interaction between domains of the death-fold superfamily. Analysis of ASC PYD mutants showed that interaction surfaces that mediate procaspase-8 interaction overlap with those required for ASC self-association and interaction with the PYDs of inflammasome initiators. Our data indicate that multiple types of death-fold domain filaments form at inflammasomes and that PYD/DED and homotypic PYD interaction modes are similar. Interestingly, we observed condensation of procaspase-8 filaments containing the catalytic domain, suggesting that procaspase-8 interactions within and/or between filaments may be involved in caspase-8 activation. Procaspase-8 filaments may also be relevant to apoptosis induced by death receptors.

Complement factor H binds to human serum apolipoprotein E and mediates complement regulation on high-density lipoprotein particles [Immunology]

October 14th, 2015 by

The alternative pathway of complement is an important part of the innate immunity response against foreign particles invading the human body. To avoid damage to host cells, it needs to be efficiently down-regulated by plasma factor H (FH) as exemplified by various diseases caused by mutations in its domains 19-20 (FH19-20) and 5-7 (FH5-7). These regions are also the main interaction sites for microbial pathogens that bind host FH to evade complement attack. We have previously shown that inhibition of FH binding by a recombinant FH5-7 construct impairs survival of FH-binding pathogens in human blood. In this study we found that, upon exposure to full blood, addition of FH5-7 reduces survival of surprisingly also those microbes that are not able to bind FH. This effect was mediated by inhibition of complement regulation and subsequently enhanced neutrophil phagocytosis by FH5-7. We found that although FH5-7 does not reduce complement regulation in the actual fluid phase of plasma it reduces regulation on HDL particles in plasma. Using affinity chromatography and mass spectrometry we revealed that FH interacts with serum apolipoprotein E (apoE) via FH5-7 domains. Furthermore, binding of FH5-7 to HDL was dependent on the concentration of apoE on the HDL-particles. These findings explain why addition of FH5-7 to plasma leads to excessive complement activation and phagocytosis of microbes in full anticoagulated blood. In conclusion, our data show how FH interacts with apoE molecules via domains 5-7, and regulates alternative pathway activation on plasma HDL particles.
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The Polycomb Repressive Complex 1 Protein BMI1 is Required for Constitutive Heterochromatin Formation and Silencing in Mammalian Somatic Cells [Genomics and Proteomics]

October 14th, 2015 by Abdouh, M., Hanna, R., El Hajjar, J., Flamier, A., Bernier, G.

The Polycomb Repressive Complex 1 (PRC1), containing the core BMI1 and RING1A/B proteins, mono-ubiquitylates histone H2A (H2Aub) and is associated with silenced developmental genes at facultative heterochromatin. It is however assumed that the PRC1 is excluded from constitutive heterochromatin in somatic cells based on work performed on mouse embryonic stem cells and oocytes. We show here that BMI1 is required for constitutive heterochromatin formation and silencing in human and mouse somatic cells. BMI1 was highly enriched at intergenic and pericentric heterochromatin, co-immunoprecipitated with the architectural heterochromatin proteins HP1, DEK1 and ATRx, and was required for their localization. In contrast, BRCA1 localization was BMI1-independent and partially redundant with that of BMI1 for H2Aub deposition, constitutive heterochromatin formation and silencing. These observations suggest a dynamic and developmentally regulated model of PRC1 occupancy at constitutive heterochromatin, and where BMI1 function in somatic cells is to stabilize the repetitive genome
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