NKT TCR recognition of CD1d-α-C-galactosylceramide

NKT cells respond to a variety of CD1d-restricted glycolipid Ags that are structurally related to the prototypic Ag α-galactosylceramide (α-GalCer). A modified analog of α-GalCer with a carbon-based glycosidic linkage (α-C-GalCer) has generated great interest because of its apparent ability to promote prolonged, Th1-biased immune responses. In this study, we report the activation of spleen NKT cells to α-C-GalCer, and related C-glycoside ligands, is weaker than that of α-GalCer. Furthermore, the Vβ8.2 and Vβ7 NKT TCR affinity for CD1d-α-C-GalCer, and some related analogs, is ～10-fold lower than that for the NKT TCR-CD1d-α-GalCer interaction. Nevertheless, the crystal structure of the Vβ8.2 NKT TCR-CD1d-α-C-GalCer complex is similar to that of the corresponding NKT TCR-CD1d-α-GalCer complex, although subtle differences at the interface provide a basis for understanding the lower affinity of the NKT TCR-CD1d-α-C-GalCer interaction. Our findings support the concept that for CD1d-restricted NKT cells, altered glycolipid ligands can promote markedly different responses while adopting similar TCR-docking topologies.     

Synthesis of glycosylated human interleukin-1α, neoglyco IL-1α, coupled with N-acetylneuraminic acid

In order to develop glycosylated cytokine, recombinant human IL-1 was chemically modified with N-acetylneuraminic acid (NANA). NANA with C9 spacer, 8-(hydrazinocarbonyl)octyl 5-acetamido-3, 5-dideoxy-D-glycero--D-galacto-2-nonulo-pyranosidonic acid potassium salt (6), was synthesized by glycosylation of C9 spacer, 8-[2-N-(benzyloxycarbonyl)hydrazinocarbonyl]octanol, with methylthio glycoside derivatives of NANA in the presence of molecular sieves 3Å and methyl(methylthio)sulfonium trifrate in propionitrile, followed by separation of and anomers with a column chromatography and deprotection. Compound 6 was coupled to IL-1 by the acyl azide method. The glycosylated IL-1 was purified by anion-exchange chromatography, and NANA coupled to IL-1 was confirmed by oxidation with NalO_4–. Based on the molecular weight average number of carbohydrate molecules introduced per molecule of IL-1 was estimated to be 2.9.     

Effects of point mutations in pVHL on the binding of HIF-1α

The von Hippel-Lindau tumor suppressor protein (pVHL) has an essential role in the regulation of the hypoxia response pathway in animal cells. Under normoxic conditions, the hypoxia-inducible factor (HIF) undergoes trans-4-prolyl hydroxylation and is subsequently recognised by the β-domain of pVHL, leading to the ubiquitination and degradation of HIF. Mutations of pVHL alter the binding of HIF. A subset of relevant clinically observed mutations to pVHL are thought to cause weaker binding of HIF-1α and are associated with cancer and cardiovascular diseases. Here, we present computational studies analyzing the interaction of HIF with mutant forms of pVHL, describing at atomic detail the local structural reorganization caused by substitution of certain residues of pVHL. The results reveal that the canonical configuration in the wild-type system is vital for the efficient functioning of the complex and that mutation of any of the residues implicated in the h-bond network in the binding site disrupts HIF binding. Although the experimentally observed ordering of binding energies for mutants of Tyr98 is reproduced, our examination of a broader range of mutations does not support the hypothesis of a correlation between the degree of disruption of the pVHL/HIF-1α interaction caused by a mutation and the phenotype with which the mutation is associated. We suggest that disruption of the binding interaction is one of many factors behind the manifestation of VHL disease.     

Synthesis of p-trifluoroacetamidophenyl O-α-D-galactopyranosyl-(1→2)-O-α-D-mannopyranosyl-(1→4)-α-L-rhamnopyranoside

The role of exposed tyrosine side-chains in enzyme-catalysed reactions has been studied for porcine-pancreatic alpha-amylase, sweet-potato beta-amylase, and Aspergillus niger glucamylase using N-acetylimidazole as the specific protein reagent. The changes in activity binding affinity (Δk_?1/k₊₁), and kinetic parameters (K_m,k₂) due to acetylation of the phenolic hydroxyl groups have been determined. Acetylation of each enzyme occurred by an “apparent” first-order reaction with a rate constant of 0.72–1.4 x 10^?1min^?1. Acetylation increased the apparent K_m (soluble starch as the substrate) for each enzyme (appreciably for alpha-amylase and glucamylase), whereas k² remained unchanged. Similarly, for each enzyme, the binding affinity for immobilised cyclohexa-amylose decreased appreciably, whereas the catalytic activity was reduced only to a small degree (and remained unchanged for beta-amylase). It is concluded that the tyrosine groups located in the active centre of each enzyme have a substrate-binding function.     

Preparation and Characterization of SDF-1α-Chitosan-Dextran Sulfate Nanoparticles

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under appropriate conditions. The glycan nanoparticles are useful carriers for protein factors, which facilitate the in vivo delivery of the proteins and sustain their retention in the targeted tissue. The glycan polyelectrolyte complexes are also ideal for protein delivery, as the incorporation is carried out in aqueous solution, which reduces the likelihood of inactivation of the proteins. Proteins with a heparin-binding site adhere to dextran sulfate readily, and are, in turn, stabilized by the binding. These particles are also less inflammatory and toxic when delivered in vivo. In the protocol described below, SDF-1α (Stromal cell-derived factor-1α), a stem cell homing factor, is first mixed and incubated with dextran sulfate. Chitosan is added to the mixture to form polyelectrolyte complexes, followed by zinc sulfate to stabilize the complexes with zinc bridges. The resultant SDF-1α-DS-CS particles are measured for size (diameter) and surface charge (zeta potential). The amount of the incorporated SDF-1α is determined, followed by measurements of its in vitro release rate and its chemotactic activity in a particle-bound form.     

Modulation of endogenous prostaglandins by thymosin-α1 in lymphocytes

The effects of thymosin-α₁ on the stimulation of specific release of prostaglandin E₂ (PGE₂) from splenic lymphocytes and thymocytes were studied. Experiments were also performed to study in parallel the absolute levels of thymosin-α₁ in the blood and the induction of serum FTS activity and of azathioprine sensitivity of spleen cells from adult thymectomized (ATx) mice. A significant difference in the release of PGE₂ between normal splenocytes and splenocytes from ATx mice was observed. Thymosin-α₁ at certain concentrations was able to stimulate PGE₂ release from lymphocytes of ATx mice while inhibiting release in lymphocytes of normal mice. Also, thymocytes were stimulated to release PGE₂ after incubation with α₁ in a manner similar to that seen in spleen cells of ATx mice. Approximately the same concentration of α₁ was found to also correct the low azathioprine sensitivity of splenocytes from ATx mice. Determinations of FTS-like activity in the blood and the pharmacokinetics of α₁ after administration of this synthetic molecule show a clear dissociation. A maximum peak of α₁ activity was obtained after 1 hr, while maximal FTS-like activity was observed after 24 hr. The inhibition of the induction by α₁ of FTS-like activity and of Thy 1.2 antigen by indomethacin suggests that the action of α₁ requires prostaglandin biosynthesis.     

α-MSH inhibits TNF-α-induced maturation of human dendritic cells in vitro through the up-regulation of ANXA1

α-Melanocyte-stimulating hormone (α-MSH), an anti-inflammatory and immunomodulatory neuropeptide, has been shown to be effective in the experimental treatment of autoimmune diseases and allograft rejection. However, its regulatory mechanism is still unclear. Mature dendritic cells (DCs) are pivotal initiators of immune response and inflammation. We hypothesized that the regulatory role of α-MSH in DC maturation would contribute to the effects of α-MSH in immune-response-mediated disease models. It was found that α-MSH inhibited tumor necrosis factor-alpha (TNF-α)-induced maturation of human peripheral-monocyte-derived DCs (MoDCs), both phenotypically and functionally. This occurred through the down-regulation of the expression of co-stimulatory molecules CD83 and CD86, the production of IL-12, the promotion of IL-10 secretion, and the MoDC phagocytic activity, suggesting that the inhibition of DC maturation by α-MSH could contribute to the anti-inflammatory effect of this neuropeptide. Furthermore, increased expression of annexin A1 (ANXA1) was found to be responsible for the α-MSH inhibiting effect on TNF-α-induced MoDC maturation, which could be abolished by the treatment of MoDCs with specific, small interfering RNAs targeting ANXA1 (ANXA1-siRNA), suggesting that α-MSH-induced ANXA1 mediates the inhibition. Therefore, α-MSH inhibits TNF-α-induced maturation of human DCs through α-MSH-up-regulated ANXA1, suggesting that inhibition of the maturation of DCs by α-MSH could mediate the anti-inflammatory effect of the neuropeptide. Furthermore, ANXA1 could be identified as a new therapeutic drug target based on the role of DCs in immune-mediated inflammatory diseases.     

Peroxiredoxin 1 Stimulates Endothelial Cell Expression of VEGF via TLR4 Dependent Activation of HIF-1α

Chronic inflammation leads to the formation of a pro-tumorigenic microenvironment that can promote tumor development, growth and differentiation through augmentation of tumor angiogenesis. Prostate cancer (CaP) risk and prognosis are adversely correlated with a number of inflammatory and angiogenic mediators, including Toll-like receptors (TLRs), NF-κB and vascular endothelial growth factor (VEGF). Peroxiredoxin 1 (Prx1) was recently identified as an endogenous ligand for TLR4 that is secreted from CaP cells and promotes inflammation. Inhibition of Prx1 by CaP cells resulted in reduced expression of VEGF, diminished tumor vasculature and retarded tumor growth. The mechanism by which Prx1 regulates VEGF expression in normoxic conditions was investigated in the current study. Our results show that incubation of mouse vascular endothelial cells with recombinant Prx1 caused increases in VEGF expression that was dependent upon TLR4 and required hypoxia inducible factor-1 (HIF-1) interaction with the VEGF promoter. The induction of VEGF was also dependent upon NF-κB; however, NF-κB interaction with the VEGF promoter was not required for Prx1 induction of VEGF suggesting that NF-κB was acting indirectly to induce VEGF expression. The results presented here show that Prx1 stimulation increased NF-κB interaction with the HIF-1α promoter, leading to enhanced promoter activity and increases in HIF-1α mRNA levels, as well as augmented HIF-1 activity that resulted in VEGF expression. Prx1 induced HIF-1 also promoted NF-κB activity, suggesting the presence of a positive feedback loop that has the potential to perpetuate Prx1 induction of angiogenesis. Strikingly, inhibition of Prx1 expression in CaP was accompanied with reduced expression of HIF-1α. The combined findings of the current study and our previous study suggest that Prx1 interaction with TLR4 promotes CaP growth potentially through chronic activation of tumor angiogenesis.     

Inhibitors of human 20α-hydroxysteroid dehydrogenase (AKR1C1)

Human 20α-hydroxysteroid dehydrogenase (AKR1C1), a member of the aldo-keto reductase (AKR) superfamily, is one of four isoforms (with >84% amino acid sequence identity) existing in human tissues. AKR1C1 most efficiently reduces biologically active progesterone and 5α-pregnan-3α-ol-20-one into their corresponding 20α-hydroxysteroids among the isoforms. The enzyme also accepts endogenous and xenobiotic non-steroidal carbonyl compounds as the substrates. In addition to the up-regulation of the AKR1C1 gene in cancer cells, the enzyme's over-expression in the cells of lung, ovary, uterine cervix, skin and colon carcinomas was reported to be associated with resistance against several anticancer agents. Thus, AKR1C1 may be a marker of the above cancers and a target of poor prognosis in cancer therapy. The recently determined X-ray crystal structures of AKR1C1/NADP(+)/20α-hydroxyprogesterone and AKR1C1/NADP(+)/3,5-dichlorosalicylic acid ternary complexes have provided a strong foundation for structure-based design methods to improve inhibitor selectivity and potency. In this review we provide an overview of the different types of AKR1C1 inhibitors and an update on the design of potent and selective inhibitors based on the crystal structure of the enzyme-inhibitor complex. Article from the Special issue on Targeted Inhibitors.     

Identification of LRP1B-interacting proteins and inhibition of protein kinase Cα-phosphorylation of LRP1B by association with PICK1

Recent studies show LDL receptor-related protein 1B, LRP1B as a transducer of extracellular signals. Here, we identify six interacting partners of the LRP1B cytoplasmic region by yeast two-hybrid screen and confirmed their in vivo binding by immunoprecipitation. One of the partners, PICK1 recognizes the C-terminus of LRP1B and LRP1. The cytoplasmic domains of LRP1B are phosphorylated by PKCα about 100 times more efficiently than LRP1. Binding of PICK1 inhibits phosphorylation of LRP1B, but does not affect LRP1 phosphorylation.This study presents the possibility that LRP1B participates in signal transduction which PICK1 may regulate by inhibiting PKCα phosphorylation of LRP1B.

Structured summary

MINT-6801075: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with SNTG2 (uniprotkb:Q925E0) by two hybrid (MI:0018)MINT-6801030, MINT-6801468: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with Pick1 (uniprotkb:Q80VC8) by two hybrid (MI:0018)MINT-6801284: LRP1B4 (uniprotkb:Q9JI18) physically interacts (MI:0218) with RanBPM (uniprotkb:P69566) by anti tag coimmunoprecipitation (MI:0007)MINT-6801108: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with Grb7 (uniprotkb:Q03160) by two hybrid (MI:0018)MINT-6801090: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with RanBPM (uniprotkb:P69566) by two hybrid (MI:0018)MINT-6801008: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with Jip-1b (uniprotkb:Q9WVI9-1) by two hybrid (MI:0018)MINT-6801052: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with Jip-2 (uniprotkb:Q9ERE9) by two hybrid (MI:0018)MINT-6801258, MINT-6801271: LRP1B4 (uniprotkb:Q9JI18) physically interacts (MI:0218) with Pick1 (uniprotkb:Q80VC8) by anti tag coimmunoprecipitation (MI:0007)MINT-6801244: RanBPM (uniprotkb:P69566) physically interacts (MI:0218) with mLRP4 (uniprotkb:Q8VI56) by anti tag coimmunoprecipitation (MI:0007)MINT-6801131, MINT-6801158: LRP1B4 (uniprotkb:Q9JI18) physically interacts (MI:0218) with Jip-1b (uniprotkb:Q9WVI9-1) by anti tag coimmunoprecipitation (MI:0007)MINT-6801231: PICK1 (uniprotkb:Q80VC8) physically interacts (MI:0218) with mLRP4 (uniprotkb:Q8VI56) by anti tag coimmunoprecipitation (MI:0007)MINT-6801173: Jip-1b (uniprotkb:Q9WVI9-1) physically interacts (MI:0218) with mLRP4 (uniprotkb:Q8VI56) by anti tag coimmunoprecipitation (MI:0007)     

Effects of Mutating Leucine to Threonine in the M2 Segment of α1 and β1 Subunits of GABAAα1β1 Receptors

The conserved leucine residues at the 9′ positions in the M2 segments of α₁ (L264) and β₁ (L259) subunits of the human GABA_A receptor were replaced with threonine. Normal or mutant α₁ subunits were co-expressed with normal or mutant β₁ subunits in Sf9 cells using the baculovirus/Sf9 expression system. Cells in which one or both subunits were mutated had a higher ``resting' chloride conductance than cells expressing wild-type α₁β₁ receptors. This chloride conductance was blocked by 10 mm penicillin, a recognized blocker of GABA_A channels, but not by bicuculline (100 μm) or picrotoxin (100 μm) which normally inhibit the chloride current activated by GABA: nor was it potentiated by pentobarbitone (100 μm). In cells expressing wild-type β₁ with mutated α₁ subunits, an additional chloride current could be elicited by GABA but the rise time and decay were slower than for wild-type α₁β₁ receptors. In cells expressing mutated β₁ subunits with wild-type or mutated α₁ subunits (αβ(L9′T) and α(L9′T)β(L9′T)), no response to GABA could be elicited: this was not due to an absence of GABA_A receptors in the plasmalemma because the cells bound [³H]-muscimol. It was concluded that in GABA_A channels containing the L9′T mutation in the β₁ subunit, GABA-binding does not cause opening of channels, and that the L9′T mutation in either or both subunits gives an open-channel state of the GABA_A receptor in the absence of ligand. Received: 17 April 1996/Revised: 5 July 1996     

Effect of 1-Methyladenine on Localization of Gαi Protein in Starfish Oocytes

Localization and quantitative dynamics of _i subunit of G protein was studied by electron immunocytochemistry and immunoenzyme assay after hormonal induction of oocyte maturation in starfish Asterias amurensis. G_i protein was chiefly localized in the plasma membrane of immature oocytes; 1-methyladenine induced redistribution of the _i protein from the plasma membrane to intracellular structure up to the breakdown of the germinal vesicle.